Etched1 protein associated with plant resistant starch, and encoding gene and application thereof

Abstract

The application discloses an Etched1 protein related to plant resistant starch, a coding gene and application thereof, and the Etched1 protein is a protein with the following amino acid sequence: 1) a protein with the amino acid sequence shown in SEQ ID NO:1. Results show that the coding gene of the Etched1 protein can increase the resistant starch content and amylose content in kernels, and change the structure of starch, thereby providing a new gene resource for corn high-resistant starch or high-amylose breeding; the coding gene of the Etched1 protein can be used for cultivating a high-resistant starch or high-amylose corn variety through molecular marker assisted breeding, gene editing breeding and a genetic engineering method, thereby providing a powerful means for improving the starch of corn seeds and improving kernel traits.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant genetic breeding and genetic engineering, and particularly relates to an Etched1 protein related to plant resistant starch, a coding gene thereof and application. BACKGROUND

[0002] Resistant starch (RS) refers to a total of starches or starch degradation products that cannot be absorbed in the small intestine of healthy individuals. Studies have shown that resistant starch has multiple physiological functions such as reducing postprandial blood glucose and improving insulin sensitivity, preventing constipation and colon cancer, maintaining intestinal health, reducing serum cholesterol and triglycerides, reducing fat accumulation, maintaining body weight, and promoting mineral absorption. As a food raw material and food additive such as a food thickening agent, a stabilizer and a modifier, resistant starch has important applications in the field of food industry; and as a medical component, such as a filler, a bandage, a decomposition substance and a thickening substance, a drug sustained-release carrier and the like, resistant starch also has important applications in the field of pharmaceutical industry.

[0003] In view of the important application value of resistant starch in the prevention and control of diabetes, if the blood glucose of diabetic patients can be controlled by increasing the content of resistant starch in diet, great benefits will be brought to diabetic patients and medical costs will be saved.

[0004] At present, most of the resistant starches are prepared from high amylose starch as raw material through a series of processing techniques, but generally the taste of such products is poor, and they are generally only used as auxiliary foods for special groups. In recent years, with the rapid development of genetic engineering, it has been proved that the cultivation of high resistant starch high-quality crop new varieties through genetic improvement is an effective strategy for dietary prevention. However, due to the unclear formation mechanism of resistant starch, so far, there are few reports on resistant starch related genes, and the determination of resistant starch is time-consuming, laborious and costly, and is easily affected by the environment, so the cultivation of high resistant starch high-quality crop new varieties faces great difficulties. Therefore, the cloning and functional research of resistant starch related genes and the development of molecular markers will greatly accelerate the process of cultivating high resistant starch high-quality crop new varieties. SUMMARY

[0005] In order to solve the technical problems in the background art, the present application provides an Etched1 protein related to plant resistant starch, a coding gene thereof and application.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] The first aspect of the present application provides an Etched1 protein related to plant resistant starch, wherein the Etched1 protein is a protein having any one of the following amino acid sequences:

[0008] 1) a protein having an amino acid sequence shown in SEQ ID NO: 1;

[0009] 2) a protein related to plant resistant starch derived from 1) by substitution and / or deletion and / or addition of one or several amino acid residues of the amino acid sequence shown in SEQ ID NO: 1.

[0010] Preferably, the several amino acid residues are no more than 10 amino acid residues.

[0011] The second aspect of the present application provides a gene encoding the above-mentioned Etchedl protein related to plant resistant starch, i.e. a DNA molecule encoding the above-mentioned Etchedl protein related to plant resistant starch, which is a DNA molecule having any one of the following:

[0012] 1) a nucleotide sequence thereof shown in SEQ ID NO: 2;

[0013] 2) a coding sequence thereof shown in SEQ ID NO: 2 from the 5' end 175th to 675th nucleotide;

[0014] 3) a coding sequence thereof shown in SEQ ID NO: 3 from the 5' end 411th to 671st nucleotide;

[0015] 4) a nucleotide sequence of genomic DNA thereof shown in SEQ ID NO: 4 from the 5' end 1st to 4306th nucleotide;

[0016] 5) a DNA molecule hybridizing to 1), 2), 3) or 4) under stringent conditions and encoding a protein having the same function;

[0017] 6) a DNA molecule having more than 90% homology to the DNA molecule of 1), 2), 3) or 4) and encoding a protein having the same function.

[0018] Preferably, the stringent conditions are hybridization at 65°C in a solution of 0.1 x SSPE (or 0.1 x SSC), 0.1% SDS and washing the membrane in a DNA or RNA hybridization experiment.

[0019] The third aspect of the present application provides primers for amplifying or synthesizing the above-mentioned Etchedl protein related to plant resistant starch full-length or any fragment thereof, and a recombinant vector, an expression cassette, a transgenic cell line, a recombinant bacterium or a transgenic cell line containing the gene encoding the above-mentioned Etchedl protein related to plant resistant starch.

[0020] Preferably, the recombinant vector is a recombinant (expression) vector constructed by inserting the DNA molecule encoding the Etchedl protein associated with plant resistant starch into an expression vector. Specifically, the recombinant (expression) vector containing the DNA molecule encoding the Etchedl protein associated with plant resistant starch can be constructed by inserting the DNA molecule encoding the Etchedl protein associated with plant resistant starch into the plant expression vector available in the art.

[0021] Preferably, the plant expression vector includes Agrobacterium binary vector and vector for plant microprojectile bombardment, etc., such as pCAMBIA1300, pCAMBIA1301, pCAMBIA3301, pCAMBIA2301, pBI121, pBin19, p*7WG2, pMDC32 or other derived plant expression vectors.

[0022] Preferably, the recombinant vector is obtained by inserting the DNA molecule encoding the Etchedl protein associated with plant resistant starch into the multiple cloning site of the plant expression vector pCAMBIA1300; the recombinant vector is an expression vector obtained by inserting the sequence 4 from the 5' end of the 1-4306thnucleotide into the Kpnl and EcoRI sites of the pCAMBIA1300 vector, named 35S:Etchedl.

[0023] Preferably, in the construction of the recombinant vector using the DNA molecule encoding the Etchedl protein associated with plant resistant starch, a promoter can be added before the transcription initiation nucleotide, which can be any one of the enhanced, constitutive, tissue-specific or inducible promoters, such as the cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin promoter and actin promoter, etc., which can be used alone or in combination with other promoters;

[0024] Preferably, in the construction of the recombinant vector using the DNA molecule encoding the Etchedl protein associated with plant resistant starch, an enhancer can also be used, which is a translation enhancer or a transcription enhancer, the translation enhancer region can be the ATG initiation codon or the adjacent region initiation codon, etc., but must be the same reading frame as the coding sequence to ensure the correct translation of the entire sequence, the source of the translation control signal and the translation initiation codon is widely available, which can be natural or synthetic; the translation initiation region can be from the transcription initiation region or the structural gene.

[0025] Preferably, in order to facilitate the screening of the transgenic plant cells or plants, the above plant expression vector can be processed, for example, by adding genes that express enzymes or luminescent compounds that can produce color changes (such as GUS genes, GFP genes, luciferase genes, etc.), antibiotic markers (such as gentamicin markers and kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes).

[0026] Preferably, the recombinant bacteria are obtained by introducing the above recombinant vector into the target bacteria, and the target bacteria are Agrobacterium tumefaciens EHA105.

[0027] The fourth aspect of the present application provides a verification method of the above-mentioned DNA molecule encoding the Etched1 protein related to the resistant starch of plants, comprising the following steps:

[0028] S1, by comparing and analyzing the resistant starch content and amylose content in the wild type W64A and Etched1 mutant grains, it is found that the resistant starch content and amylose content in the Etched1 mutant grains are significantly higher than those in the wild type, which indicates that the mutant gene Etched1 is related to the resistant starch and amylose content;

[0029] S2, by cloning the Etched1 gene through PCR technology and constructing a complementary vector, the Etched1 mutant is transformed by the Agrobacterium transformation method, and the resistant starch content and amylose content in the grains of the wild type W64A, the Etched1 mutant and the transgenic material are compared and analyzed. The resistant starch content and amylose content in the grains of the transgenic material return to the level of the wild type W64A. At the same time, by constructing the Etched1 gene antisense vector and transforming it into the wild type W64A, the resistant starch content and amylose content in the grains of the W64A transformed with the Etched1 gene antisense vector are significantly increased, which indicates that the Etched1 gene is involved in the regulation of the resistant starch content and amylose content.

[0030] The fifth aspect of the present application provides the use of the Etched1 protein associated with plant resistant starch, the DNA molecule encoding the Etched1 protein associated with plant resistant starch, the recombinant vector containing the DNA molecule encoding the Etched1 protein associated with plant resistant starch, the expression cassette, the transgenic cell line and the recombinant bacteria in regulating the grain traits of plants, which is characterized by the increase of resistant starch content, and / or the increase of amylose content, and / or the change of starch chain length distribution; wherein the increase of resistant starch content and amylose content is achieved by functional loss of the Etched1 protein coding gene, which is caused by any one of the mutations of insertion, conversion, transversion, frameshift or deletion of the Etched1 protein coding gene; the plant is a dicotyledon or a monocotyledon, and is further preferably corn.

[0031] The sixth aspect of the present application provides the use of the Etched1 protein associated with plant resistant starch, the DNA molecule encoding the Etched1 protein associated with plant resistant starch, the recombinant vector containing the DNA molecule encoding the Etched1 protein associated with plant resistant starch, the expression cassette, the transgenic cell line and the recombinant bacteria in preparing the products of cultivating high resistant starch plants and / or high amylose plants; the products of cultivating the high resistant starch plants and / or high amylose plants are prepared by molecular marker assisted breeding, gene editing breeding and genetic engineering method; the plant is a monocotyledon or a dicotyledon; and the monocotyledon is specifically corn.

[0032] The seventh aspect of the present application provides a method for cultivating a transgenic plant, which comprises the following steps: introducing a DNA molecule encoding the Etched1 protein associated with plant resistant starch into a target plant, or using a gene editing method to change the Etched1 gene sequence of the target plant, to obtain a transgenic plant; the transgenic plant has the following C1) and / or C2) and / or C3) characteristics:

[0033] C1) the resistant starch content of the grain of the transgenic plant is higher than that of the target plant;

[0034] C2) the amylose content of the grain of the transgenic plant is higher than that of the target plant;

[0035] C3) the starch chain length distribution of the grain of the transgenic plant is changed;

[0036] The target plant (transformed plant host) is a dicotyledon or a monocotyledon, and is further preferably corn, and more preferably W64A.

[0037] Preferably, the DNA molecule encoding the Etched1 protein related to plant resistant starch is introduced into the target plant by a plant expression recombinant vector; the plant expression recombinant vector containing the DNA molecule encoding the Etched1 protein related to plant resistant starch is transformed into plant cells or tissues by a biological method such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, etc.

[0038] The eighth aspect of the present application provides a method for regulating plant grain traits including resistant starch content, and / or amylose content and / or starch property structure, which comprises: introducing the above-mentioned DNA molecule encoding the Etched1 protein related to plant resistant starch into a recipient plant; the recipient plant is a dicotyledon or monocotyledon.

[0039] The present application has the following advantages:

[0040] (1) The present application separates a gene capable of regulating resistant starch content and / or amylose content by the method of allelic mutation, and verifies the function of the gene through transgenic functional complementation experiment;

[0041] (2) In the Etched1 homozygous mutant, the expression amount of Etched1 is significantly reduced, the resistant starch content and amylose content of the grain are greatly improved, and the chain length distribution of starch is also changed;

[0042] (3) After the coding gene of the Etched1 protein is introduced into the Etched1 homozygous mutant, the phenotype of the mutant can be complemented, and the resistant starch content and amylose content in the grain of the complemented transgenic plant return to the wild type level;

[0043] (4) After the activity of the coding gene of the Etched1 protein is reduced in corn, the resistant starch content and amylose content in the corn grain are greatly improved, and the chain length distribution of starch is also changed.

[0044] In summary, the present application provides the Etched1 protein related to plant resistant starch, its coding gene and application. The experimental results show that the coding gene of the Etched1 protein can increase the resistant starch content and amylose content in the grain, and change the structure of starch, thereby providing a new gene resource for high resistant starch or high amylose breeding of corn; according to the coding gene of the Etched1 protein and its mutant, a primer sequence can be designed to carry out molecular marker assisted breeding, gene editing breeding and high resistant starch or high amylose corn variety breeding by using genetic engineering method, which provides a powerful means for starch improvement of corn seeds and grain trait improvement, and has important theoretical significance and great application potential. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1 The results of Etched1 genotyping in wild-type W64A and Etched1 allelic mutants;

[0047] Figure 2 The content of resistant starch and amylose in the grains of wild-type W64A and Etched1 allelic mutants:

[0048] A: Resistant starch content in grains of wild-type W64A and Etched1 allelic mutants;

[0049] B: Amylose content in the grains of wild-type W64A and Etched1 allelic mutants;

[0050] Figure 3 The results of the expression analysis of the Etched1 gene;

[0051] Figure 4 The results show the resistant starch and amylose content in transgenic maize kernels during a functional complementation experiment; among them,

[0052] A: Schematic diagram of the construction of the gEtched1-et1 complementary vector;

[0053] B: Expression level of Etched1 in transgenic maize as detected by real-time quantitative PCR;

[0054] C: The content of resistant starch in complementary transgenic corn kernels; D: The content of amylose in complementary transgenic corn kernels;

[0055] (W64A is the non-transgenic control, gEtched1-et1 is the transgenic plant, and EV-et1 is the empty vector transgenic control plant; T-test, lowercase letters represent significant (P<0.05), uppercase letters represent highly significant (P<0.01; same letters represent no difference, different letters represent a difference).

[0056] Figure 5 The content of resistant starch and amylose in transgenic maize kernels during RNA interference experiments, among which,

[0057] A: The expression of Etchedl in transgenic maize detected by real-time fluorescent quantitative PCR;

[0058] B: The content of resistant starch in transgenic maize grain by RNA interference;

[0059] C: The content of amylose in transgenic maize grain by RNA interference;

[0060] W64A is non-transgenic control, RNAi-W64A is transgenic plant, EV-W64A empty vector transgenic control plant;

[0061] (T test, lower case letters represent significant (P <0.05), upper case letters represent extremely significant (P <0.01), the same letters represent no difference, different letters represent difference). DETAILED DESCRIPTION

[0062] In the following description, for the sake of explanation, but not for limitation, specific details are set forth such as particular techniques, structures, etc. in order to provide a thorough understanding of embodiments of the present application. However, it will be obvious to those skilled in the art that the present application can be practiced in other embodiments that depart from these specific details.

[0063] Example 1

[0064] 1. Determination and obtaining of target gene

[0065] The seeds of Zea mays L. Etchedl allele mutant etl and W64A (see Figure 1 ) were cultivated respectively, and the specific cultivation method was as follows: the corn seeds were soaked in 30°C water for 6 hours, the water surface was 1-2 centimeters higher than the seeds, the seeds were filtered and dried after soaking, and single grain was planted in the greenhouse. Then the leaf was taken to extract DNA.

[0066] 1) Extraction of corn genomic DNA:

[0067] The improved CTAB method was used to extract genomic DNA from rice leaves. 100 mg of rice leaves were frozen with liquid nitrogen, ground into powder in a 5 cm diameter mortar, transferred to a 1.5 mL centrifuge tube to extract DNA, and the obtained DNA precipitate was dissolved in 100 μL MQ H2O.

[0068] 2) Identification of mutant genotype

[0069] The genotype of et1 and W64A was identified by using EtCAPs 1F / 1R primer combination in Table 1. After the PCR amplification with EtCAPs F / R primer combination using et1 genomic DNA as template, and then after the MspA1I enzyme digestion, two bands of 150bp and 82bp were obtained by gel electrophoresis, while the product after the PCR amplification with EtCAPs F / R primer combination using W64A genomic DNA as template, and then after the MspA1I enzyme digestion, the product could not be cut open because there was no MspA1I enzyme digestion site, and then by gel electrophoresis, a band of 232bp was obtained, as shown in Figure 1. Figure 1 The results show that et1 is a homozygous mutant.

[0070] Table 1.

[0071]

[0072] 3) Identification of mutant phenotype

[0073] According to the resistant starch method of American Association of Analytical Chemists (AOAC) (2002.02), the resistant starch in the wild type W64A and mutant et1 was determined, and the results are shown in et1 A. According to the amylose determination method reported by Perez and Juliano (1978), the amylose content in the wild type W64A and mutant et1 grains was determined, and the results are shown in Figure 2 B. Figure 2

[0074] Figure 2 A results show that, et1 The resistant starch content in the grain (11.63%) was significantly higher than that in the wild type W64A grain (6.7%).

[0075] Figure 2 B results show that, et1 The amylose content in the grain (27.84%) was significantly higher than that in the wild type W64A grain (24.61%).

[0076] 4) Expression level of resistant starch related gene Etched1 in W64A and et1 young spikes

[0077] ​Ten days after pollination, young ears were harvested and immediately frozen in liquid nitrogen and stored at -80°C until use. Total RNA was extracted from young ears using Trizol (Invitrogen) and treated with DNase (Invitrogen). One μg of total RNA was used to synthesize cDNA with M-MLV reverse transcriptase (Promega) according to the manufacturer's instructions. Real-time quantitative PCR was performed using the qPCR primer combinations in Table 3, SYbGreen mix (Bio-Rad) and a fluorescence quantitative PCR instrument (7500, ABI) according to the manufacturer's instructions. The reaction program was: 50°C, 2 min; 95°C, 10 min; then 95°C, 15 seconds, 60°C, 1 min, 40 cycles. The ubiquitin carrier protein (UBCP) gene was used as a reference gene. The results are shown in Figure 3 .

[0078] From Figure 3 The results show that the expression level of Etched1 in et1 young ears is about 66% of that in wild type W64A.

[0079] Example 2

[0080] 1. Obtaining and detecting transgenic plants

[0081] 1.1. Construction of recombinant expression vector

[0082] 1.1.1. Cloning of genes

[0083] A DNA fragment containing the Etched1 gene was amplified from W64A genomic DNA using the primer combination gEtched11F / gEtched11R in Table 2 (the nucleotide sequence of the preliminary fragment is shown in SEQ ID NO: 4). The preliminary fragment was digested with Kpnl and EcoRI and recovered to obtain a final genomic DNA fragment containing the full-length Etched1 (the nucleotide sequence of the fragment is shown in SEQ ID NO: 4 from the 1st to the 4306th nucleotide at the 5' end Figure 4 A).

[0084] The nucleotide sequence of the cDNA corresponding to the genomic DNA shown in SEQ ID NO: 4 from 1 to 4306bp is shown in SEQ ID NO: 2 or SEQ ID NO: 3. Sequence 2 consists of 875 bases, and its open reading frame (ORF) is from the 175th to the 675th base from the 5' end, encoding an Etchedl protein having the amino acid sequence shown in SEQ ID NO: 1. Sequence 3 consists of 833 bases, and its open reading frame (ORF) is from the 411th to the 671st base from the 5' end, encoding an isoform of the Etchedl protein having the amino acid sequence shown in SEQ ID NO: 1. The 5' end of the gEtchedl IF primer is added with a Kpnl enzyme cutting site, and the 5' end of the gEtchedl IR primer is added with an EcoRI enzyme cutting site.

[0085] Table 2. Sequences of the gEtchedl IF primer and the gEtchedl IR primer

[0086]

[0087] 1.1.2. Construction of a recombinant expression vector

[0088] The obtained final genomic DNA fragment containing the full-length Etchedl gene was inserted into the Kpnl and Sbfl enzyme cutting sites of the vector pCAMBIA1300 (purchased from the Cambia Company), to obtain a recombinant expression vector gEtchedl (see Figure 1). Figure 4 A).

[0089] 1.1.3. Obtaining of transgenic plants

[0090] The plasmid gEtchedl was transformed into Agrobacterium by electroporation, and a recombinant Agrobacterium strain containing the recombinant plasmid gEtchedl was screened. The young spikes of etl 10 days after pollination were sterilized by removing the bracts, and the young embryos were peeled off and washed in the infection culture solution (Methods in Molecular Biology, Vol. 343: Agrobacterium Protocols, 2 / e, volume 1) for one to two times, for standby use.

[0091] The recombinant Agrobacterium strain containing the recombinant plasmid gEtchedl was used to infect the gEtchedl maize immature embryos. Then the immature embryos were co-cultured in co-cultivation medium (Methods in Molecular Biology, vol.343: Agrobacterium Protocols, 2 / e, volume 1) for 3 days in dark at 20°C, and then the immature embryos were transferred to selection medium containing 50 mg / L hygromycin (Methods in Molecular Biology, vol.343: Agrobacterium Protocols, 2 / e, volume 1) to screen the resistant callus and transgenic plants. The hygromycin resistant plants were transplanted into greenhouse or field after acclimatization in shade, and the obtained transgenic plants were T0 generation. The transgenic seeds of T0 generation were planted to obtain the gEtchedl transformed T1 generation transgenic plants.

[0092] According to the method of obtaining gEtchedl transformed T1 generation transgenic plants, the empty vector pCAMBIA1300 was transformed into etl to obtain empty vector control plants.

[0093] 2. Detection of transgenic plants

[0094] 1) Detection of Etchedl gene expression by real-time fluorescent quantitative PCR

[0095] The total plant RNA of transgenic plants and control plants W64A was extracted by Trizol (purchased from Invitrogen Company), and reverse transcription was performed by reverse transcription kit (purchased from Promega Company) to obtain cDNA. The primers qEtchedl F and qEtchedl were used to detect the expression of Etchedl gene by PCR. Ubiquitin carrier protein gene was amplified by qUBCP F and qUBCP R as an internal standard. The primer sequences are shown in Table 3, and the results are shown in Figure 4 B.

[0096] Table 3. Primer sequences

[0097]

[0098] Figure 4 B. The results show that the expression of Etchedl gene in transgenic plants is increased.

[0099] 2) Detection of resistant starch content and amylose content of transgenic plants

[0100] The resistant starch content and amylose content in the seeds of the T1 generation transgenic plants of gEtched1, W64A wild type plants and empty vector control plants were determined. The results are shown in Table 2. Figure 4 C and Figure 4 D, respectively,

[0101] From Figure 4 C and Figure 4 D, it can be seen that the resistant starch content and amylose content in the seeds of the T1 generation transgenic plants of gEtched1 were decreased compared with the empty vector control, and there was no significant difference compared with the W64A wild type plants.

[0102] Example 3

[0103] Obtaining and detecting of transgenic plants

[0104] 1. Obtaining of transgenic plants

[0105] 1) Obtaining of interference fragments

[0106] The W64A maize young ears were collected at 10 days after pollination, immediately frozen in liquid nitrogen, and then stored in a -80°C refrigerator. After total RNA was extracted from the young ears by the Trizol method (purchased from Invitrogen Corporation), DNase (purchased from Promega Corporation) was added for treatment. According to the manufacturer's instructions, 1 μg of total RNA was used to synthesize cDNA by M-MLV reverse transcriptase (Promega Corporation).

[0107] The above young ear cDNA was subjected to PCR amplification by primers RNAi 1F / RNAi 1R and RNAi 2F / RNAi 2R in Table 4, respectively, and the obtained products were sequenced. The nucleotide sequences of the gene fragments amplified by the two pairs of primers are SEQ ID NO: 5 and SEQ ID NO: 6, respectively. SEQ ID NO: 5 is a fragment of 3bp to 662bp of SEQ ID NO: 2, and SEQ ID NO: 6 is the reverse complement sequence of SEQ ID NO: 4. SEQ ID NO: 4 and SEQ ID NO: 5 were confirmed by whole genome alignment analysis to have no other homologous sequences in the maize genome. The 5' ends of the primers of the RNAi were added with gateway vector recombination adapters, respectively.

[0108] Table 4, primer sequences

[0109]

[0110] 2) Construction of interference vectors

[0111] The product amplified by the primer pair RNAi1F / RNAi1R was inserted into the Gateway vector p*7GWIWG2(II) with 35S promoter by Gateway vector system recombination to obtain the recombinant expression vector Etched1-RNAi (i.e. interference vector Etched1-RNAi), and the inserted fragment forms a hairpin structure after expression.

[0112] 3) Obtaining of transgenic plants

[0113] The interference vector Etched1-RNAi was transformed into Agrobacterium tumefaciens strain EHA105 by electroporation, and a recombinant Agrobacterium strain containing the interference vector Etched1-RNAi was screened. The young ears of W64A 10 days after pollination were sterilized by removing the bracts, and the young embryos were peeled off and washed in infection culture solution (Methods in Molecular Biology, Vol. 343: Agrobacterium Protocols, 2 / e, volume 1) for one to two times for standby.

[0114] After the recombinant Agrobacterium strain containing the interference vector Etched1-RNAi was used to infect the W64A corn young embryos, the young embryos were placed on co-culture medium (Methods in Molecular Biology, vol. 343: Agrobacterium Protocols, 2 / e, volume 1), and the young embryos without Agrobacterium transformation were used as a control. The young embryos were co-cultured at 20°C in the dark for 3 days, and then transferred to a selection medium containing 50 mg / L hygromycin for screening of resistant callus and transgenic plants. The hygromycin-resistant plants were hardened in a cool place, and then transplanted into a field to obtain T0 generation transgenic plants. The T0 generation transgenic seeds were planted to obtain T1 generation transgenic plants of Etched1-RNAi.

[0115] According to the method of obtaining T1 generation transgenic plants of Etched1-RNAi, the empty vector p*7GWIWG2(II) was used to transform W64A to obtain empty vector control plants.

[0116] 2, Detection of transgenic plants

[0117] 1) Detection of expression amount of Etched1 by real-time fluorescent quantitative PCR

[0118] The total plant RNA of the transgenic plants and the control plants W64A was extracted by Trizol (purchased from Invitrogen Corporation), and reverse transcription was performed by using a reverse transcription kit (purchased from Promega Corporation) to obtain cDNA. The expression amount of the Etched1 gene was detected by real-time fluorescent quantitative PCR by using primers qEtched11F and qEtched11R in Table 3. The Ubiquitin carrier protein gene was amplified by using qUBCP F and qUBCP R as an internal standard, and the primer sequences are shown in Table 3. The results are shown in Figure 5 A.

[0119] Figure 5 The results show that the expression amount of Etched1 in the transgenic plants is reduced.

[0120] 2) Detection of resistant starch content and amylose content of the transgenic plants

[0121] The resistant starch content and amylose content in the seeds of the T1 generation of the transgenic plants of Etched1-RNAi, the W64A control plants and the empty vector control plants were determined. The results are shown in 5B and Figure 5 C.

[0122] From the results of Figure 5 B and Figure 5 C, compared with the W64A control plants and the empty vector control plants, the resistant starch content and the amylose content in the seeds of the T1 generation of the transgenic plants of Etched1-RNAi are both greatly increased, in which the resistant starch is increased from 6.7% to 10.6%, and the amylose is increased from 24.6% to 26.9%.

[0123] The present application is not limited to the above specific embodiments, and various modifications made by those skilled in the art without creative labor based on the above concept all fall within the protection scope of the present application.

Claims

1. A method of breeding a transgenic maize plant, comprising, The method comprises the following steps: changing the Etched1 gene sequence of the corn by a gene editing method, specifically, gene function loss caused by any one of the mutations of insertion, conversion, transversion, frame shift or deletion of the Etched1 protein coding gene, to obtain the transgenic corn; the transgenic corn has the following C1) and / or C2) characteristics: C1) the resistant starch content of the transgenic corn grain is higher than that of the corn; C2) the amylose content of the transgenic corn grain is higher than that of the corn.

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