A method for increasing plant seeds and / or plants

Abstract

The present invention discloses a method for increasing plant seeds and / or plants, which is characterized in that the expression of genes encoding proteins ST1 and ST2 in plants is reduced or removed by gene interference or gene knockout methods. The present invention for the first time reveals the biological functions of the Arabidopsis thaliana ST1 and ST2 genes working together. Mutants with reduced or non-expression of the ST1 and ST2 genes cause the seeds of Arabidopsis thaliana to increase in size and the mature plants to increase in size. The present invention has important implications for plant breeding.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for increasing plant seeds and / or plants. Background Art

[0002] Seed size affects the evolutionary adaptability and stress resistance of plants and is a key factor directly determining plant yield. Revealing the genetic and molecular mechanisms of seed size regulation is not only an important developmental biology issue but also has important theoretical significance and application value for increasing seed yield.

[0003] The size of seeds is jointly regulated by the external growth environment and the plant's own genetic information. Among them, the endogenous genetic information in maternal tissues, zygotic embryos, and endosperm is the main factor for seed size regulation. Seeds usually consist of three parts: embryo, endosperm, and seed coat. The size of seeds is closely related to the coordinated growth of these three parts. After double fertilization, endosperm growth precedes embryo growth, and the division of endosperm cells leads to a rapid increase in seed volume. It has been found that some factors in Arabidopsis can regulate seed size by affecting endosperm growth. When the embryo develops to the globular embryo stage, its volume only accounts for a very small part, and the seed size is already close to the final size. Therefore, the endosperm plays a crucial role in determining seed size. The maternal tissue integument develops into the seed coat later, providing space for the growth of the embryo and endosperm and determining the size of the seed. Therefore, during the process of maternal tissue regulating seed size development, the genotype of the maternal tissue rather than the genotype of the zygote determines the size of the seed.

[0004] In the studies of Arabidopsis and rice, pathways for maternal tissue to regulate seed size have been reported, including the ubiquitin proteasome pathway, G-protein signaling pathway, hormone signaling pathway, and transcription factor-mediated pathway, etc. In addition to the pathways related to endosperm, embryo, and maternal tissue regulation that affect seed size development, the MAPK signaling pathway has also been found to play an important role in regulating seed development ([1]Li N,Li Y(2016)Signaling pathways of seed size control in plants.Curr Opin Plant Biol 33:23–32.[2]Li N,Xu R,Li Y(2019)Molecular Networks of Seed Size Control in Plants.Annu Rev Plant Biol 70:435–463).

[0005] Seed size is regulated by complex developmental and environmental signals. Although some key seed size regulatory genes and some signaling pathways have been identified through research, our understanding of this regulatory network remains limited. Therefore, further exploration and discovery of genes that affect seed size are of great significance for understanding the process of seed development and improving crop yields. Summary of the Invention

[0006] The present invention provides a method for increasing the size of plant seeds and / or plants. The present inventors found that when the expression levels of two genes, ST1 and ST2, are reduced or not expressed, the seeds of Arabidopsis thaliana can be enlarged and the mature plants can be increased in size.

[0007] A method for increasing the size of plant seeds and / or plants, by reducing or eliminating the expression of genes encoding proteins ST1 and ST2 in plants through gene interference or gene knockout methods.

[0008] Preferably, the plant is a dicotyledonous plant. More preferably, the plant is Arabidopsis thaliana.

[0009] Among them, the coding regions of the genes encoding proteins ST1 and ST2 are shown as SEQ ID No.5 and SEQ ID No.6, respectively. Further, the gene sequences of the genes encoding proteins ST1 and ST2 are shown as SEQ ID No.3 and SEQ ID No.4, respectively, and these two sequences contain some non-coding regions.

[0010] In the specific operation of the method described above, the genes encoding proteins ST1 and ST2 are respectively subjected to gene interference to reduce expression, or respectively subjected to gene knockout to eliminate expression, or one of the genes is subjected to interference to reduce expression and the other gene is subjected to gene knockout to eliminate expression. Two single-gene interference or knockout plants of the two genes can be constructed first, and then the two single-gene interference or knockout plants are hybridized to screen for plants in which both genes are interfered or knocked out.

[0011] The present invention also provides the application of the genes encoding proteins ST1 and ST2 in increasing the size of plant seeds and / or plants, by reducing or eliminating the expression of the genes encoding proteins ST1 and ST2 in plants through gene interference or gene knockout methods.

[0012] The present invention for the first time reveals the biological functions of the Arabidopsis thaliana ST1 and ST2 genes acting together. Mutants with reduced or non-expressed ST1 and ST2 genes have enlarged seeds and increased mature plants in Arabidopsis thaliana. The present invention has important implications for plant breeding. Brief Description of the Drawings

[0013] Figure 1 It is a diagram of the T-DNA insertion positions of st1-1, st1-2, st2-1, and st2-2 mutants.

[0014] Figure 2 Electrophoresis detection chart for detecting gene expression levels in st1-2 and st2-1 homozygous mutants.

[0015] Figure 3 Growth phenotype chart of st1-2 st2-1, st1-1 st2-1, st1-1 st2-2 homozygous double mutants and Col-0 plants at the mature stage.

[0016] Figure 4 Statistical chart of seed grain length and width of st1-2 st2-1, st1-1 st2-1, st1-1 st2-2 homozygous double mutants and Col-0 plants. Error bars represent the standard error. * indicates a significant difference compared with Col-0. *P<0.05, ***P<0.001, ****P<0.0001.

[0017] Figure 5 Statistical chart of 1000-seed weight of st1-2 st2-1, st1-1 st2-1, st1-1 st2-2 homozygous double mutants and Col-0 plants. Error bars represent the standard error. * indicates a significant difference compared with Col-0. ****P<0.0001. Detailed implementation methods

[0018] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of this invention.

[0019] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific implementation methods of this invention's specification, which are obvious to those skilled in the art. Other implementation methods obtained from this invention's specification are obvious to those skilled in the art. This invention's specification and examples are merely exemplary.

[0020] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0021] The methods used in the following examples are all conventional methods unless otherwise specified.

[0022] Plant materials:

[0023] The wild type of Arabidopsis thaliana is the Columbia ecotype Col-0.

[0024] The ST1 gene mutant st1-1 was purchased from the ABRC (Arabidopsis Biological Resource Centre) seed center, and the product catalog number is SAIL_1253_B02.

[0025] The ST1 gene mutant st1-2 was purchased from the ABRC (Arabidopsis Biological Resource Centre) seed center, and the product catalog number is SALK_066477.54.35.x.

[0026] The ST2 gene mutant st2-1 was purchased from the ABRC (Arabidopsis Biological Resource Centre) seed center, and the product catalog number is SALK_069738.42.15.x.

[0027] The ST2 gene mutant st2-2 was purchased from the ABRC (Arabidopsis Biological Resource Centre) seed center, and the product catalog number is SAIL_315_C08.

[0028] Compared with the genomes of mutants st1-1, st1-2 and wild-type Arabidopsis Col-0, only the ST1 gene has T-DNA insertion mutations, and the rest of the genes are the same. The T-DNA is inserted into the first exon and promoter respectively ( Figure 1 ).

[0029] Compared with the genomes of mutants st2-1, st2-2 and wild-type Arabidopsis Col-0, only the ST2 gene has T-DNA insertion mutations, and the rest of the genes are the same. The T-DNA is inserted into the first intron and 3’UTR respectively ( Figure 1 ).

[0030] The nucleotide sequence of the ST1 gene is SEQ ID No.3, the amino acid sequence of the protein it encodes is SEQ ID No.1, and the nucleotide sequence of the coding region is as shown in SEQ ID No.5.

[0031] The nucleotide sequence of the ST2 gene is SEQ ID No.4, the amino acid sequence of the protein it encodes is SEQ ID No.2, and the nucleotide sequence of the coding region is as shown in SEQ ID No.6.

[0032] Example 1

[0033] Application of Arabidopsis ST1 and ST2 genes in jointly regulating plant seed size and mature plant size.

[0034] I. Identification of st1 and st2 single mutant materials

[0035] 1. Plant st1-1, st1-2, st2-1 and st2-2 on 1 / 2 MS medium. After growing for 2 weeks, extract the plant genome and identify the genotype. The mutant genotype was identified by the three-primer method. The primers were designed by the T-DNA primer design website (http: / / signal.salk.edu / tdnaprimers.2.html). The primers designed by the website were denoted as st1-2-LP and st1-2-RP, st2-1-LP and st2-1-RP, and the LB primer was LBb1.3 provided by the website. st1-1-LP and st1-1-RP, st2-2-LP and st2-2-RP, and the LB primer was LB1 provided by the website.

[0036] The primer sequences are as follows:

[0037] st1-2-LP: 5’-TCCCATATTCTCCGTCTACCC-3’;

[0038] st1-2-RP: 5’-ATCAAAGCAGATACACCACCG-3’;

[0039] st2-1-LP: 5’-GACCAATACACATATATACGCAGC-3’;

[0040] st2-1-RP: 5’-CATGGTCCGGTATAGGTAAACCG-3’;

[0041] LBb1.3: 5’-ATTTTGCCGATTTCGGAAC-3’;

[0042] st1-1-LP: 5’-ATCCAATCCTCTGAAACCCTC-3’;

[0043] st1-1-RP: 5’-GTGCTTACAGGAGTTGCTTCG-3’;

[0044] st2-2-LP: 5’-CTTACCGTTTGATCGTGATCG-3’;

[0045] st2-2-RP: 5’-TGTTTTGATGCAATATCGTGTG-3’;

[0046] LB1: 5’-GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC-3’.

[0047] The PCR experiment was carried out using the 2×Rapid Taq Master Mix (Vazyme, P222) kit. The PCR reaction conditions were as follows:

[0048] Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 58°C for 15 sec, extension at 72°C for 15 sec, for a total of 32 cycles; extension at 72°C for 5 min.

[0049] Plants with a homozygous genotype identification were retained and transplanted into nutrient soil.

[0050] 2. Widhalm et al. (Widhalm JR, Ducluzeau AL, Buller NE, Elowsky CG, Olsen LJ, Basset GJC (2012) Phylloquinone (vitamin K1) biosynthesis in plants: Two peroxisomal thioesterases of lactobacillales origin hydrolyze 1,4-dihydroxy-2-naphthoyl-coa. Plant J 71:205–215) have identified that st1-1 and st2-2 are mutants with reduced expression levels. After 2 weeks, the RNA of st1-2 and st2-1 was extracted, and semi-quantitative expression level detection was performed after reverse transcription. The primers for detecting the expression level of the ST1 gene were ST1-RT-F and ST1-RT-R, the primers for detecting the expression level of the ST2 gene were ST2-RT-F and ST2-RT-R, and the primers for detecting the expression level of the internal reference gene UBC9 were UBC9-RT-F and UBC9-RT-R.

[0051] The primer sequences were as follows:

[0052] ST1-RT-F: 5’-TGGATTCTGCATCGTCCAACA-3’;

[0053] ST1-RT-R: 5’-GTTTGCTGCATCTTTGGCGT-3’;

[0054] ST2-RT-F: 5’-TATTCGATGAGCTCTCCGCC-3’;

[0055] ST2-RT-R: 5’-TTCAGCTCGTCTGGAGCATC-3’;

[0056] UBC9-RT-F: 5’-CCGTTGCGGAAGACATGTTTCATT-3’;

[0057] UBC9-RT-R: 5'-TAGGGCTCTTCCTTAAGGACAGTA-3'.

[0058] The PCR reaction conditions for the UBC9 primer were as follows:

[0059] Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 58°C for 15 sec, extension at 72°C for 15 sec, for a total of 28 cycles; extension at 72°C for 5 min.

[0060] The PCR reaction conditions for the ST1 and ST2 primers were as follows:

[0061] Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 58°C for 15 sec, extension at 72°C for 15 sec, for a total of 30 cycles; extension at 72°C for 5 min.

[0062] The semi-quantitative PCR results of st1-2 and st2-1 indicated that st1-2 and st2-1 were gene knockout mutants ( Figure 2 ).

[0063] All plants were harvested for seeds individually after maturity.

[0064] II. Preparation of the st1st2 double mutant material

[0065] Plant the homozygous mutant plants of st1-1, st1-2, st2-1, and st2-2. During the flowering period, remove the open flowers on the inflorescence, select the unopened flowers for emasculation, use forceps to remove the sepals, petals, and filaments to expose the stigma. Additionally, pick the flowers that opened on the same day from the open inflorescence, remove the style, and use the anthers on the filaments to pollinate the stigmas of the emasculated flowers of another mutant. Repeat the pollination multiple times. Wrap the artificially pollinated style with a moist plastic wrap, remove the plastic wrap after two or three days, and observe the growth of the siliques.

[0066] Harvest the seeds separately from the successfully pollinated and growing siliques. After drying, plant them in 1 / 2 MS medium. After growing for 2 weeks, extract the plant genome and identify the genotypes of the offspring.

[0067] Retain the plants with genotypes identified as heterozygous for st1-1, st1-2, st2-1, and st2-2, i.e., obtain the heterozygous double mutant plants of st1-2 st2-1, st1-1 st2-1, and st1-1 st2-2. Transplant them into nutrient soil and harvest the seeds individually after maturity.

[0068] Plant the seeds in 1 / 2 MS medium. After growing for 2 weeks, extract the plant genome and conduct genotype identification. The primers and PCR reaction conditions are the same as above.

[0069] Retain the plants with homozygous genotypes of st1-2 and st2-1 in the offspring of st1-2 st2-1; retain the plants with homozygous genotypes of st1-1 and st2-1 in the offspring of st1-1 st2-1; retain the plants with homozygous genotypes of st1-1 and st2-2 in the offspring of st1-1 st2-2, that is, obtain the st1-2 st2-1, st1-1 st2-1, and st1-1 st2-2 homozygous double mutant plants. Transplant them into nutrient soil and harvest the seeds from each individual plant when mature.

[0070] III. Phenotype of st1st2 double mutant

[0071] Plant Col-0 and the st1-2 st2-1, st1-1 st2-1, and st1-1 st2-2 homozygous double mutant materials simultaneously under the same growth conditions. When the plants are mature and the rosette leaves stop growing and the plants reach their maximum height, take pictures of the plants. The results show that the rosette leaf area of the st1-2 st2-1, st1-1 st2-1, and st1-1 st2-2 lines is significantly larger than that of Col-0, and the plant height is significantly higher than that of Col-0( Figure 3 ).

[0072] When the seeds are mature, harvest the seeds from each individual plant. After taking pictures of the seeds under a stereomicroscope, use ImageJ software to measure the length and width of each seed, and measure 350 seeds for each plant. Weigh the 1000-seed weight of Col-0 and the st1-2 st2-1, st1-1 st2-1, and st1-1 st2-2 seeds using an analytical balance. The results show that the length of the seeds of the st1-2 st2-1, st1-1 st2-1, and st1-1 st2-2 lines is significantly higher than that of Col-0, and the width has no significant difference from that of Col-0( Figure 4 ). The 1000-seed weight of the seeds of the st1-2 st2-1, st1-1 st2-1, and st1-1 st2-2 lines is significantly greater than that of Col-0( Figure 5 ).

[0073] The above results indicate that when the expression level of st1-2 st2-1 is reduced or even not expressed, the size (area) of the seeds can be increased, the 1000-seed weight can be increased, and the rosette leaf area of the mature plants and the height of the mature plants can be increased.

Claims

1. A method for increasing the size of plant seeds and / or increasing the rosette leaf area of mature plants and increasing the height of mature plants, characterized in that, Reduce or eliminate the expression of the genes encoding proteins ST1 and ST2 in plants by gene interference or gene knockout methods; The gene sequences encoding proteins ST1 and ST2 are shown as SEQ ID No.3 and SEQ ID No.4 respectively; The plant is Arabidopsis thaliana.

2. The method according to claim 1, wherein Reduce the expression of the genes encoding proteins ST1 and ST2 by gene interference respectively, or eliminate the expression by gene knockout respectively, or reduce the expression of one gene by interference and eliminate the expression of the other gene by gene knockout.

3. The method according to claim 2, wherein First, construct single-gene interference or knockout plants for the two genes respectively, and then hybridize the two single-gene interference or knockout plants to screen and obtain plants with interference or knockout of both genes.

4. Use of genes encoding proteins ST1 and ST2 in increasing plant seeds and / or increasing the rosette leaf area of mature plants and increasing the height of mature plants, characterized in that, Reduce or eliminate the expression of the genes encoding proteins ST1 and ST2 in plants by gene interference or gene knockout methods; The gene sequences encoding proteins ST1 and ST2 are shown as SEQ ID No.3 and SEQ ID No.4 respectively; The plant is Arabidopsis thaliana.

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