Gene, biological material and method for regulating histidine content in crops

Abstract

The invention provides a gene, a biological material and a method for regulating and controlling histidine content in crops, and belongs to the technical field of gene engineering. The invention provides a TaATL15 gene for regulating and controlling the histidine content in crops, and the amino acid sequence of protein coded by the TaATL15 gene is shown as SEQ ID No.3. The TaATL15 gene is transformed into wheat through a genetic transformation method by utilizing the TaATL15 gene, so that a TaATL15 overexpression transgenic plant is obtained, and the histidine content in the crops is regulated and controlled. And the contents of histidine, tyrosine and levodopa in the TaATL15 overexpression transgenic plant are obviously increased.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a gene for regulating histidine content in crops, biological materials and methods. BACKGROUND

[0002] Histidine is a semi-essential amino acid for human body. It is essential for infants and also required from food for adults under certain conditions, such as rapid tissue renewal and high metabolic state. Its unique imidazole side chain structure endows it with multiple physiological functions, enabling it to participate in important life processes such as metal ion homeostasis regulation, buffer system maintenance, immune regulation and nerve conduction in vivo. The isoelectric point of imidazole group is close to physiological pH, which makes it have strong buffering capacity in blood. Secondly, histidine is a component of hemoglobin, myoglobin and the active center of various enzymes, can bind with metal ions such as iron, zinc and copper, participate in enzymatic reactions and metal transport, and maintain metal ion balance. Moreover, histidine is the precursor molecule for synthesizing histamine, an important physiological mediator, which has the effects of promoting smooth muscle contraction, microvascular dilation, gastric acid secretion, etc. Therefore, histidine can lower blood pressure and is clinically used for the treatment of diseases such as angina pectoris and heart failure. It can be seen that histidine has important significance for maintaining human health.

[0003] For plants, histidine not only participates in the synthesis of plant proteins as a structural unit, but also plays a key role in various metabolic and physiological processes. Recent studies have shown that histidine has multiple functions in plants, and it can participate in various metabolic reactions and has a significant impact on plant nutrient absorption and utilization efficiency. In addition, histidine shows important physiological significance in metal ion chelation and transport, stress response, etc. In the aspect of metal ion metabolism, histidine forms complexes with metal ions such as Ni 2+ , Zn 2+ , etc., and participates in their transport and detoxification in plants.

[0004] Amino acid transporters can be divided into amino acid-polyamine-choline transporter superfamily (APC) and amino acid / auxin permease superfamily (AAAP). The AAAP superfamily is mainly responsible for the one-way active transport (proton cooperation) of amino acids, including amino acid permease (AAP), lysine-histidine transporter (LHT), proline transporter (ProT), gamma-aminobutyric acid transporter (GAT), auxin transporter (AUX), aromatic and neutral amino acid transporter (ANT), amino acid transporter-like (ATL). A variety of amino acid transporters have been reported in plants such as rice and Arabidopsis. Different types of amino acid transporters in these reports will affect the content of different types of amino acids in plants, so it is particularly important to explore wheat amino acid transporter related genes to specifically improve the content of different amino acids in wheat. SUMMARY

[0005] The application provides a gene, a biological material and a method for regulating the content of histidine in crops, and the gene can increase the content of histidine in crops.

[0006] The application provides a TaATL15 gene for regulating the content of histidine in crops, and the amino acid sequence of the protein encoded by the TaATL15 gene is shown in SEQ ID No. 3.

[0007] In a preferred mode of the application, the full-length genomic nucleotide sequence of the TaATL15 gene is shown in SEQ ID No. 2.

[0008] In a preferred mode of the application, the CDS nucleotide sequence of the TaATL15 gene is shown in SEQ ID No. 1.

[0009] The application also provides a primer set for amplifying the above-mentioned TaATL15 gene.

[0010] In a preferred mode of the application, the TaATL15-F is shown in SEQ ID No. 4 and the TaATL15-R is shown in SEQ ID No. 5.

[0011] The application also provides a biological material for regulating the content of histidine in crops, wherein the biological material contains the above-mentioned TaATL15 gene.

[0012] The application also provides a biological material for increasing the content of histidine in crops, comprising a recombinant vector, a recombinant strain or a recombinant crop overexpressing the above-mentioned TaATL15 gene.

[0013] The application also provides the above-mentioned TaATL15 gene, the above-mentioned biological material or the above-mentioned biological material in the regulation of the content of histidine in crops.

[0014] In a preferred mode of the present application, the regulation comprises increasing the amino acid content of the crops after overexpression of the TaATL15 gene.

[0015] The present application also provides the use of the above-mentioned TaATL15 gene, the above-mentioned biological material or the above-mentioned biological material in the breeding of crops with high histidine content.

[0016] Beneficial effects: The present application provides a TaATL15 gene for regulating the histidine content in crops, the amino acid sequence of the protein encoded by the TaATL15 gene is shown in SEQ ID No. 3, the TaATL15 gene is highly homologous to the reported OsATL15 and OsATL13 in rice, and belongs to the ATLb subfamily, and the expression of OsATL13 can increase and promote the transport of phenylalanine and methionine, while OsATL15 has no research showing that it has amino acid transport activity. By using the TaATL15 gene of the present application, the TaATL15 gene is transformed into the wheat variety Fielder by genetic transformation method, and TaATL15 transgenic plants are obtained. Through the analysis of the gene expression amount of the TaATL15 overexpression transgenic material and the content of histidine, tyrosine and levodopa in wheat leaves and grains, the results show that compared with the control wild type Fielder, the expression amount of TaATL15 gene in the grain of the positive transformation plant overexpressing TaATL15 for 3 weeks is greatly improved, and the contents of histidine, tyrosine and levodopa are significantly increased. The results of the leaves in the booting stage are consistent with those of the grains for 3 weeks, which proves that the TaATL15 gene has the functions of transporting histidine, tyrosine and dopa. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Figure for the result of genome-wide association analysis of histidine content in wheat to locate TaATL15;

[0018] Figure 2 Sequence alignment of TaATL15 and homologous genes;

[0019] Figure 3 Phylogenetic tree and expression pattern of TaATL15 protein homologous sequence analysis;

[0020] Figure 4 Difference in TaATL15 gene expression amount and histidine content in the grains of wild type wheat and transgenic wheat overexpressing TaATL15; wherein ** represents P<0.01, *** represents P<0.001, and **** represents P<0.0001;

[0021] Figure 5Differences in TaATL15 gene expression and histidine content in the shoots and leaves of wild-type wheat and transgenic wheat overexpressing TaATL15; wherein ** represents P<0.01, *** represents P<0.001, **** represents P<0.0001;

[0022] Figure 6 Structure diagram of a genetic transformation vector for overexpressing TaATL15 in wheat. DETAILED DESCRIPTION

[0023] The present application provides a TaATL15 gene for regulating the content of histidine in crops, wherein the amino acid sequence of the encoded protein of the TaATL15 gene is shown in SEQ ID No. 3.

[0024] The amino acid sequence of the encoded protein of the TaATL15 gene is shown in SEQ ID No. 3: MEDHTTSRLAATASPLGEPLLTATGQRADRDAEAQLPPYRSAGASFSRTCLNLTNAVSGIGVLTMPYAVSRGGWLSLALFALVGAVCYYTGTLMARCMRADGSVATYPDIGQLAFGAAGRRTVAVFMYVELYLVAISFLVLEGDNLDKLFPGAGVELLPGYRLRGKQLFIVLAGAVVLPTTWLRNLGVLAYVSALGLVASAALTASLVWAGVSESGFRTNGNVLNLAGLPASLGLYFVCFTGHVVFPTIYSSMKNRERFSQVLLISSVLCGLNYGFTAVLGYLIYGEDVQSQVTLNLPSGRLYTKIAIVMTLINPLTKYALLVAPITSAIEERFSLMGSGPARVAVSTAVLVSTVAVACMVPFFGFLMSFIGSFLSVMATVFFPCLCYLKIYKAKGLRRVEVAAIVGILLLGAFVAITGTYTSLLDIIGTF*.

[0025]

[0026]

[0027] The application also provides a primer set for amplifying the TaATL15 gene.

[0028] The primer set comprises TaATL15-F as shown in SEQ ID No. 4 and TaATL15-R as shown in SEQ ID No. 5.

[0029] TaATL15-F (SEQ ID No. 4): 5'-ATGGAAGACCACACGACGAG-3';

[0030] TaATL15-R (SEQ ID No. 5): 5'-TCAGAAAGTGCCTATGATGTCCAA-3'.

[0031] When the primer set is used for amplification, cDNA obtained by reverse transcription of total RNA extracted from crops is used as a template, and TaATL15-F and TaATL15-R are used as a primer pair for amplification. The amplification system is 15 μL, including: DNA (20 ng / ul) 2 μL, Forward primer (5 uM) 1 μL, Reverse primer (5 uM) 1 μL, 2X PCR mixture 7.5 μL, and ddH2O 3.5 μL. After the PCR amplification system is configured, PCR amplification is performed. The PCR amplification program comprises 94℃ 5 min; 94℃ 30 s, 58℃ 30 s, 72℃ 1 min 30 s, 34 cycles; 72℃ 5 min, and 25℃ 1 min.

[0032] The application also provides a biological material for regulating the histidine content of crops, wherein the biological material contains the TaATL15 gene.

[0033] In an embodiment of the application, the amplified fragment is subjected to a homologous recombination reaction with a target vector to obtain a recombinant vector capable of overexpressing the TaATL15 gene. In an embodiment, pLGY is selected as the target vector.

[0034] The application also provides a biological material for increasing the histidine content of crops, comprising a recombinant vector, a recombinant strain or a recombinant crop overexpressing the TaATL15 gene.

[0035] In an embodiment of the application, the recombinant vector has Figure 6The recombinant Agrobacterium containing the recombinant vector is obtained by transforming the recombinant vector into Agrobacterium after the vector map is shown, and finally the genetic transformation is carried out by using the Agrobacterium-mediated genetic transformation system to obtain the recombinant crop with overexpressed TaATL15 gene. The crop of the present application is not particularly limited, which can be monocotyledonous plants, Poaceae plants, Poinae plants, and Triticum plants such as wheat.

[0036] The present application also provides the application of the above-mentioned TaATL15 gene, the above-mentioned biological material or the above-mentioned biological material in regulating the histidine content of crops.

[0037] The regulation of the present application includes increasing the amino acid content of crops after overexpressing the TaATL15 gene.

[0038] The present application also provides the application of the above-mentioned TaATL15 gene, the above-mentioned biological material or the above-mentioned biological material in breeding crops with high histidine content.

[0039] The method for breeding crops of the present application is not particularly limited, for example, in one embodiment, the method provided in the prior art (Hayta S, Smedley MA, Demir S U, et al. An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat (Triticum aestivum L.) [J]. Plant Methods, 2019, 15: 1-15.) is used, the genetic transformation is completed by Agrobacterium EHA105 mediation and wheat genetic transformation system, and the breeding of crops is completed.

[0040] In order to further illustrate the present application, the gene, biological material and method for regulating the histidine content of crops provided by the present application are described in detail in combination with examples below, but they should not be understood as limiting the scope of protection of the present application.

[0041] Example 1: Obtaining of TaATL15 gene

[0042] 1. Determination and structural analysis of TaATL15 gene

[0043] The inventors used HPLC-MS / MS technology to detect the free histidine content of wheat natural population materials, and located a SNP site (SNP-124839279) on chromosome 4A which was significantly associated with the zinc content in wheat grains. Figure 1), and the gene annotation is lysine-histidine transporter, the gene number is TraesCS4A01G018500, and the name is TaATL15. The markers used in GWAS analysis are all referred to the IGWSC1.0 version of the reference genome https: / / urgi.versailles.inra.fr / download / iwgsc / IWGSC_RefSeq_Assemblies / v1.0 / iwgs c_refseqv1.0_all_chromosomes.zip.

[0044] The full-length genomic sequence of TaATL15 is 1296 bp (SEQ ID No. 1), the gene has one exon, the CDS sequence is 990 bp (SEQ ID No. 2), and it encodes 330 amino acids (SEQ ID No. 3).

[0045] The sequence alignment analysis of the AAAP family in rice and Arabidopsis and TaATL15 is carried out Figure 2 ), and the phylogenetic tree analysis Figure 3 ) shows that TaATL15 is highly homologous to the reported OsATL15 and OsATL13 in rice, and belongs to the ATLb subfamily.

[0046] 2, Amplification of TaATL15 gene

[0047] The total RNA of the wheat variety Zhonghuanchun is extracted, and the cDNA is reversely transcribed, and the specific primers TaATL15-F and TaATL15-R are used for amplification. The PCR amplified target fragment is connected with a blunt-ended vector, and the reaction product is transformed into E. coli DH5α, 1 mL of LB is recovered for 1 h, 150 μL of the recovered bacterial liquid is coated on the LA medium containing resistance AMP, and the plate is cultured in a 37°C incubator for 12 h. The single colony is detected by PCR, and the plasmid is sequenced. The PCR product is sequenced and analyzed, and the cDNA sequence of TaATL15 gene is obtained. The 1296 bp TaATL15 gene sequence amplified from Zhonghuanchun is obtained. After sequence alignment, homologous recombination is carried out with the target vector.

[0048] Example 2: Regulation of TaATL15 genetic material on wheat histidine content

[0049] 1, Construction of TaATL15 genetic transformation vector

[0050] The 1296 bp TaATL15 gene fragment amplified from Zhonghuanchun in Example 1 is subjected to homologous recombination reaction with the target vector pLGY, and the enzyme digestion site is BamHI, to obtain the recombinant vector, and the vector map is shown in Figure 6The vector is an Agrobacterium-mediated genetic transformation vector carrying a maize ubiquitin gene promoter with constitutive and overexpression characteristics.

[0051] 2. TaATL15 genetic transformation

[0052] Referring to the prior art (Hayta S, Smedley MA, Demir SU, et al. An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat (Triticum aestivum L.) [J]. Plant Methods, 2019, 15: 1-15.), the plasmid correctly cloned in step 1 was introduced into the wheat variety Fielder through Agrobacterium EHA105 mediation and wheat genetic transformation system. After pre-culture, infection, co-cultivation, screening of callus tissue with glufosinate resistance, differentiation, rooting, seedling hardening and transplanting, transgenic plants were obtained.

[0053] 3. Analysis of gene expression levels in TaATL15 overexpressing transgenic materials and the contents of histidine, tyrosine, and L-dopa in wheat leaves and grains

[0054] The DNA level of T1 transgenic wheat plants was tested to confirm the construction of TaATL15 positive plants. RNA samples and metabolic samples were collected from leaves at the emergence stage and seeds 3 weeks after filling from T1 transgenic wheat plants overexpressing TaATL15 and wild-type wheat plants to analyze the expression level of TaATL15 and the content of histidine and tyrosine. The results showed that compared with the wild-type control Fielder, the expression level of the TaATL15 gene in the grains 3 weeks after filling of the positive transformed plants overexpressing TaATL15 was significantly increased ( Figure 4 A), the levels of histidine, tyrosine and levodopa increased significantly ( Figure 4 Middle B), the leaf results at the emergence stage are consistent with the grain results 3 weeks after filling ( Figure 5 ).

[0055] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A TaATL15 gene for regulating the histidine content in crops, characterized in that: The amino acid sequence of the protein encoded by the TaATL15 gene is shown in SEQ ID No.

3.

2. The TaATL15 gene according to claim 1, characterized in that The full-length genomic nucleotide sequence of the TaATL15 gene is shown in SEQ ID No.

2.

3. The TaATL15 gene according to claim 2 or 3, characterized in that The CDS nucleotide sequence of the TaATL15 gene is shown in SEQ ID No.

1. A primer set for amplifying the TaATL15 gene according to any one of claims 1 to 3.

5. The primer set according to claim 4, characterized in that The nucleotide sequences include TaATL15-F shown in SEQ ID No. 4 and TaATL15-R shown in SEQ ID No.

5.

6. A biological material for regulating the histidine content of crops, characterized in that: The biological material contains the TaATL15 gene according to any one of claims 1 to 3.

7. A biological material for increasing the histidine content in crops, characterized in that: The invention comprises a recombinant vector, a recombinant strain or a recombinant crop that overexpresses the TaATL15 gene according to any one of claims 1 to 3.

8. Use of the TaATL15 gene according to any one of claims 1 to 3, the biological material according to claim 6, or the biological material according to claim 7 in regulating the histidine content in crops.

9. The application according to claim 8, characterized in that: The regulation includes increasing the amino acid content of crops after overexpressing the TaATL15 gene.

10. Use of the TaATL15 gene according to any one of claims 1 to 3, the biomaterial according to claim 6, or the biomaterial according to claim 7 in breeding crops with high histidine content.

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