CN110759979A - Transcription factor bZIP2 for improving starch synthesis of wheat grains and application thereof - Google Patents

Abstract

The invention discloses a transcription factor bZIP2 capable of improving wheat grain starch synthesis and application thereof. The transcription factor is a TubZIP2 gene, and three copies of the TubZIP2 gene homologous gene TabZIP2 in common wheat: TabZIP-A2 , TabZIP - B2 and TabZIP-D2 , its coding region is composed of nucleotide sequences shown in SEQ ID No.1, No.2, No.3 and No.4 respectively. In TubZIP2 -overexpressing wheat lines, the expressions of AGPL-cyto, AGPS- cyto and SBEIIa were significantly increased in the middle and late stages of endosperm filling compared with those of the wild type, while the expression levels of other starch synthesis-related genes (SSRGs) did not change. Length and thousand kernel weight also increased significantly. When the gene TabZIP2 in common wheat was knocked out, the total starch and amylopectin contents of the knockout lines were significantly reduced, and the grain length and thousand-grain weight were also significantly reduced. Plant height, effective tillering, number of spikelets and grain length of TubZIP2 overexpression lines and TabZIP2 knockout lines did not change significantly.

Description

A transcription factor bZIP2 that enhances starch synthesis in wheat grains and its application

technical field

The invention relates to the field of genetic engineering, in particular to a transcription factor for improving the expression of wheat starch synthesis-related genes and its application.

Background technique

As one of the major crops in the world, wheat not only provides protein, vitamins and dietary fiber for human health, but also is the main source of starch and energy in the human diet. Starch is the main storage component in the endosperm of wheat grains, accounting for 60-70% of the total weight of the grain and 70-85% of the total weight of the flour.

Starch synthesis in wheat endosperm cells involves a series of complex reactions that require the participation of a series of enzymes. At the beginning of starch synthesis, AGPase catalyzes the conversion of glucose-1-phosphate (G1c-1-P) and adenosine triphosphate (ATP) to adenosine diphosphate glucose (ADP-glucose), which is the glucose supply for amylose and amylopectin synthesis. body. Afterwards, granule-bound starch synthase (GBSS) catalyzes the final formation of amylose from ADP-glucose, while starch synthase (SS), starch branching enzyme (SBE) and debranching enzyme (DBE) catalyze the transfer of ADP-glucose to highly branched The branched chain of glucan is finally formed into amylopectin. By regulating the expression of genes related to starch synthesis, the starch content of grains can be effectively increased, thereby increasing the yield of wheat. However, to date, there are not many reports on the transcriptional regulation of starch synthesis in cereal grains. Among them, RSR1 and OsbZIP58 are starch synthesis regulators identified in rice; ZmNAC36, ZmbZIP91 , ZmEREB156 and SUSIBA2 are transcription factors identified in maize and barley that regulate starch synthesis, respectively. In wheat, except for the study on the rice RSR1 homolog TaRSR1 , no systematic study on the regulatory mechanism of starch synthesis has been reported.

SUMMARY OF THE INVENTION

The invention identifies and obtains a transcription factor TubZIP2 that is co-expressed with starch synthesis related genes (SSRG) in the endosperm filling stage through co-expression analysis (Weighted Gene Co-expression Network Analysis, WGCNA). TubZIP2 and its homologous gene TabZIP2 in common wheat can significantly increase the expression of AGPL-cyto , AGPS-cyto and SBEIIa genes, thereby increasing the total starch and amylopectin content of wheat grains. Meanwhile, TubZIP2 and TabZIP2 did not have any adverse effects on other important agronomic traits of wheat.

The invention contributes to people's understanding of the regulation of wheat starch synthesis, and TubZIP2 and TabZIP2 can also be used as important target genes in genetic engineering and traditional breeding to cultivate high-yield and high-quality new wheat varieties.

By analyzing the transcriptome data of Uraltu wheat G1812 grain-filling endosperm, the inventors identified that TubZIP2 and some SSRG genes have similar expression patterns. TubZIP2 is subcellularly located in the nucleus, and its 1-128aa has transcriptional activation activity. TubZIP2 promoted the activity of AGPL-cyto, AGPS-cyto and SBEIIa gene promoters in dual-luciferase reporter gene system assays and transient overexpression assays in wheat endosperm. In TubZIP2 -overexpressing wheat lines, the expressions of AGPL-cyto, AGPS-cyto and SBEIIa were significantly increased in the middle and late stages of endosperm filling compared with those of the wild type, while the expression levels of other SSRG genes did not change. Correspondingly, the total starch content and amylopectin content of the overexpression lines were significantly increased, and the grain length and thousand-grain weight of the overexpression lines were also significantly increased. On the contrary, using CRISPR/Cas9 technology to knock out the homologous gene of TubZIP2 in common wheat, TabZIP2 , the total starch content and amylopectin content of the knockout line were significantly reduced, and the grain length and thousand-grain weight were also significantly reduced. Plant height, effective tillering, number of spikelets and grain length of TubZIP2 overexpression lines and TabZIP2 knockout lines were not significantly changed. In conclusion, TubZIP2 and TabZIP2 promoted wheat grain starch synthesis without negatively affecting other important agronomic traits.

The purpose of the present invention is to provide a transcription factor TubZIP2 that regulates the expression of SSRG gene, and the transcription factor promotes the synthesis of starch.

The transcription factor TubZIP2 provided by the present invention is derived from Uraltu wheat, and has a coding region composed of the nucleotide sequence shown in SEQ ID No. 1. The DNA shown in SEQ ID No.1 has a length of 1722 bp (excluding stop codons), belongs to the bZIP family, and encodes a protein of 574 amino acid residues. The homologous gene of TubZIP2 in common wheat is TabZIP2 , which has three copies of TabZIP-A2 , TabZIP-B2 and TabZIP-D2 , and their coding regions are SEQ ID No.2, SEQID No.3 and SEQ ID No.4 respectively. The nucleotide sequences are shown, and their coding regions are all 1722 bp in length, encoding a protein of 574 amino acid residues.

Meanwhile, the present invention provides a recombinant vector comprising the transcription factor of claim 1.

The present invention also provides an expression vector containing the transcription factor, preferably a plant expression vector.

The present invention provides the application of the transcription factor in improving the expression of SSRG gene at grain filling stage in wheat; the application is to transform the transcription factor into wheat, and screen positive transgenic lines overexpressing the transcription factor.

The present invention also provides a method for overexpressing the transcription factor in the endosperm. The method is to transform the overexpression vector containing the transcription factor into the endosperm of common wheat, preferably, 15 days after the flower of common wheat is transformed by a gene gun method. of endosperm.

Meanwhile, the present invention provides a universal primer pair for amplifying the full-length coding regions of TubZIP2 and TabZIP2 , wherein the sequence of the forward primer is shown in SEQ ID No. 5, and the sequence of the reverse primer is shown in SEQ ID No. 6.

The present invention also provides a method for determining candidate transcription factors for regulating SSRG gene expression by co-expression analysis, which analyzes the transcriptome of Uraltu wheat endosperm at grain filling stage by RNA-Seq, and finds transcription factors with similar expression patterns to SSRG gene.

The present invention provides a method for detecting the regulation strength of transcription factor on SSRG gene promoter region. The overexpression vector of transcription factor and the reporter vector whose SSRG gene promoter drives the expression of Firefly luciferase gene are co-transformed into wheat protoplast cells, And Renilla luciferase was used as an internal reference to detect the intensity of transcription factors regulating SSRG gene promoter activity.

The invention provides a method for detecting the regulation of SSRG gene expression by a transcription factor through endosperm overexpression. The overexpression vector of the transcription factor is transformed into the endosperm 15 days after the flower of common wheat by the method of gene gun, and cultured in a hypertonic medium for 48 hours in the dark , the changes of SSR gene expression in endosperm were detected by RT-PCR.

The Uraltu wheat TubZIP2 provided by the invention can up-regulate the expression of AGPL - cyto , AGPS - cyto and SBEIIa genes in the immature seeds of common wheat and finally increase the content of total starch and amylopectin in mature seeds. Its homologous gene in common wheat, TabZIP2, has the same function. Therefore, bZIP2 is of great significance for the improvement of starch quality in wheat.

Description of drawings

Fig. 1 TubZIP2 phylogenetic tree analysis, subcellular localization, transcriptional activation activity and expression pattern analysis. Among them, A is the structure of TubZIP2, of which 128-179 amino acids (amino acids, aa) represent the core region of bZIP family transcription factors; B is the phylogenetic tree analysis showing that TubZIP2 and its homologous genes are conserved in Triticeae, and are similar to other genes from Poaceae The homologous genes of the groups are far away; C means that the N-terminal (1-127 aa) of TubZIP2 has transcriptional activation activity; D means that TubZIP2 is located in the nucleus; E means that TubZIP2 is specifically expressed in the mature endosperm of T. urartu ; F means that TubZIP2 and some SSRG genes were co-expressed in T. urartu during grain filling.

Figure 2 TubZIP2 increases the transcription of AGPL-cyto , AGPS-cyto and SBEIIa genes in vitro. A is a schematic diagram of the reporter vector, expression vector and internal reference in the dual-luciferase reporter gene system, wherein REN: Renilla luciferase; LUC: Firefly luciferase; Ubi: ubiquitin; P: promoter; ter: termination. B is that TubZIP2 significantly improves the promoter activity of AGPL-cyto , AGPS-cyto and SBEIIa genes in the dual-luciferase reporter gene system; C is the schematic diagram of TubZIP2 overexpression vector in endosperm; D is that the overexpression of TubZIP2 in endosperm improves the Transcription of AGPL-cyto , AGPS-cyto and SBEIIa genes.

Figure 3 TubZIP2 increases the transcription levels of AGPL-cyto , AGPS-cyto and SBEIIa genes in wheat overexpression lines. A and B represent RNA-seq and RT-PCR results, respectively, showing that TubZIP2 was overexpressed in immature endosperm of transgenic wheat; C, E, and G represent endosperm at 7, 14, and 21 days after flowering, respectively, in RNA-seq analysis Changes of AGPL-cyto , AGPS-cyto and SBEIIa transcript levels in the middle; D, F and H represent RT-PCR verification of AGPL- cyto , AGPS-cyto and SBEIIa gene expression in endosperm 7 days, 14 days and 21 days after flowering, respectively quantity changes.

Figure 4 TubZIP2 increases the total starch content and amylopectin content of the overexpressed lines. A: TubZIP2 significantly increased the total starch content of the overexpression lines planted in Beijing, Dishang and Zhaoxian from 2017 to 2018; B, the electron microscope scanning comparison found that the A-type starch granules in the grains of the overexpression lines planted in Zhaoxian were more than those of the wild type, where A and B represent A- and B-type starch granules, respectively; C, D, and E represent the amylopectin content, amylose content and the ratio of amylose to amylopectin in the overexpression lines at the three sites, respectively; F is the Beijing overexpressed lines had higher peak viscosity compared to wild type in RVA analysis.

Fig. 5 TubZIP2 increases the 1000-grain weight and grain width of overexpression lines. A and B are the plant and panicle morphology of the wild type and overexpression lines, respectively; C, D and E are the plant height, effective tillers and spikelets per panicle of the wild type and overexpression lines, respectively; F is the wild type and overexpression lines The grain length and grain width morphology of the lines; G means that TubZIP2 has no effect on the grain length of transgenic lines; H and I mean that TubZIP2 increases the grain width and 1000-grain weight of the overexpression lines, respectively.

Figure 6 TabZIP2 increases the transcription of AGPL-cyto , AGPS-cyto and SBEIIa genes in vitro. A is the specific expression of TabZIP2 in the endosperm of common wheat; B is the co-expression of TabZIP2 with AGPL-cyto , AGPS-cyto and SBEIIa ; C is the schematic diagram of the overexpression vector of TabZIP2 in the endosperm; D is the overexpression of TabZIP2 in the endosperm to improve AGPL- Transcription of cyto , AGPS- cyto and SBEIIa genes.

Figure 7 Total starch content and amylopectin content decreased in TabZIP2 knockout lines. A shows that the total starch content of the TabZIP2 knockout lines in Beijing, Dishang and Zhaoxian was significantly reduced; B, it was found that the type A starch grains in the grains of the knockout lines planted in Zhaoxian were less than those of the wild type, and A and B were Indicates type A and type B starch granules; C is the amylopectin content of the knockout lines at the three sites; D is the rapid viscosity analyzer (RVA) analysis of the Beijing-grown knockout lines with lower peaks compared to the wild type Viscosity; E and F show a significant decrease in kernel width and thousand kernel weight, respectively, of the knockout lines.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1 Evolutionary tree analysis, subcellular localization, transcriptional activation activity and co-expression analysis of Uraltu wheat TubZIP2 and SSR genes, including the following steps:

(1) Cloning of wheat bZIP2 gene: According to the sequences of the 5' and 3' ends of TRIUR3_00571, primers for amplifying the coding region of TubZIP2 were designed (the sequence of the forward primer is shown in SEQ ID No. 5, and the sequence of the reverse primer is shown in SEQ ID No. 5). SEQ ID No. 6), using the 15-day post-flowering endosperm cDNA of Uraltu wheat as a template, TubZIP2 was cloned to obtain TubZIP2 , the sequence is shown in SEQ ID No. 1. Search IWGSC BLAST against TubZIP2 sequences (https://urgi.versailles.inra.fr/blast_iwgsc/ dbgroup=wheat_iwgsc_refseq_v1_chromosomes&program=blastn) and WheatExp BLAST (https://wheat.pw.usda.gov/WheatExp/), using the full-length primers of TubZIP2 to the 15-day post-flowering endosperm cDNA of common wheat as a template, the full-length TubZIP2 The primers (the sequence of the forward primer is shown in SEQ ID No. 5, and the sequence of the reverse primer is shown in SEQ ID No. 6) PCR amplification and monoclonal sequencing to obtain three copies of common wheat TabZIP2 TabZIP-A2 , TabZIP- B2 and TabZIP-D2 , their sequences are shown in SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, respectively.

(2) Phylogenetic tree analysis: The amino acid sequences of TubZIP2 , TabZIP2 and GenBank genes with more than 80% sequence similarity to them were analyzed by Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) software After the alignment, Mega 5.05 (http://www.megasoftware.net/) was used to construct an evolutionary tree. The results are shown in Figure 1B. TubZIP2 and its homologous genes in Triticaceae were clustered into a single branch in the evolutionary tree analysis. , which is far from homologous genes from other groups, which indicates that the bZIP2 gene is conserved in Triticeae.

PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome）比对，分析发现TubZIP2是一个bZIP类家族的转录因子，包含一个basicleucine zipper（bZIP）结构域（128-179 aa）结果如图1A所示，TubZIP2的128- 179 aa为它的DNA结合区域 (bZIP domain )，。将TubZIP2的全长和片段1-127 aa、128-179 aa以及180-574 aa对应的编码区序列分别与pGBKT7（Clontech，California，USA）构建重组质粒。将重组质粒分别转化酵母菌株AH109（Clontech，California，USA）结果如图1C所示，所有菌株都可以在固体培养基 (SD/-Trp)上正常生长，但只有转化完整基因和转录激活区域重组质粒的菌株可以使X-gal变蓝，而转化DNA结合区域重组质粒的菌株不能变蓝。这表明TubZIP2具有转录激活活性，且其N端1-127 aa行使转录激活活性功能。(3) Detection of transcriptional activation activity: The amino acid sequence of TubZIP2 was analyzed by NCBI Protein BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi

PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) alignment, analysis found that TubZIP2 is a bZIP family transcription factor, containing a basicleucine zipper (bZIP) domain (128-179 aa) The results are shown in Figure 1A, TubZIP2 128-179 aa is its DNA binding region (bZIP domain). The full-length TubZIP2 and the corresponding coding region sequences of fragments 1-127 aa, 128-179 aa and 180-574 aa were respectively combined with pGBKT7 (Clontech, California, USA) to construct recombinant plasmids. The recombinant plasmids were respectively transformed into yeast strain AH109 (Clontech, California, USA). The results are shown in Figure 1C. All strains could grow normally on solid medium (SD/-Trp), but only the transformed intact gene and transcriptional activation region were recombined. Strains with plasmids can turn X-gal blue, while strains transformed with recombinant plasmids in the DNA binding region cannot. This indicates that TubZIP2 has transcriptional activation activity, and its N-terminal 1-127 aa functions as transcriptional activation activity.

(4) Subcellular localization: Connect TubZIP2 to the pJIT163-UBI-hGFP vector, construct a recombinant plasmid pJIT163-UBI-TubZIP2-hGFP with TubZIP2 fused to GFP, transform wheat leaf protoplast cells, and realize TubZIP2 fusion under the drive of ubiquitin promoter. Express. As a result, as shown in Figure 1D, TubZIP2 localized to the nucleus of protoplast cells. The localization of TubZIP2 in the nucleus indicates that it functions in the nucleus, which is consistent with the characteristics of transcription factors.

(5) Tissue-specific expression analysis: Trizol method was used to extract total RNA from roots, stems, leaves and endosperm of Uraltu wheat 15 days after flowering, and TubZIP2- specific primers were used to verify their tissue-specific expression by RT-PCR. The results are as follows As shown in Figure 1E, TubZIP2 is abundantly expressed in the endosperm.

(6) Verification of the expression patterns of wheat bZIP2 and SSRG genes: In the endosperm of Uraltu wheat at grain filling stage, specific primers for TubZIP2 and some SSRG genes were used to verify the expression patterns of both by RT-PCR. As shown in Figure 1F, the expression of TubZIP2 and selected SSRG genes peaked at 5 days after flowering and then gradually decreased, which was consistent with the results of RNA-Seq.

Example 2 Tissue-specific expression of common wheat TabZIP2 and SSRG genes and in vitro promotion of transcription of AGPL-cyto , AGPS-cyto and SBEIIa .

(1) Tissue-specific expression analysis: Total RNA was extracted from the roots, stems, flag leaves, ears and endosperm of common wheat plants 15 days after Chinese spring flowering, using specific primers for TabZIP-A2 , TabZIP-B2 and TabZIP-D2 , through RT-PCR verified their tissue-specific expression, and the results were shown in Figure 6A. TabZIP-B2 was slightly expressed, and TabZIP-A2 and TabZIP-D2 had a small amount of expression in roots, stems, flag leaves, and panicles 15 days after flowering. In the endosperm 15 days after flowering, TabZIP-A2 , TabZIP-B2 and TabZIP-D2 were all expressed in a large amount, and TabZIP-B2 had the highest expression.

(2) Verification of the expression patterns of TabZIP2 and SSRG genes: RT-PCR was used to detect the expression patterns of TabZIP2 and SSRG genes in the endosperm of common wheat at the Chinese spring filling stage. The results are shown in Figure 6B. TabZIP-A2 , TabZIP-B2 and TabZIP-D2 has the same expression pattern as some SSRG genes, that is, it starts at 5 days after flowering, peaks at 10 days after flowering, and then the expression begins to decline; this also indicates that these three copies are involved in common wheat SSRG gene expression regulation.

Example 3 Function of wheat bZIP2 gene

1. Direct regulation of SSRG gene by wheat bZIP2

(1) The dual-luciferase reporter gene system was used to detect the regulation of TubZIP2 on the SSR gene promoter: The ubiquitin promoter was used to drive the massive expression of TubZIP2 (Ubi P::TubZIP2), and the expression vector of TubZIP2 was constructed; the promoter of the SSRG gene (- 2000 bp to -1 bp ) to drive the expression of Firefly luciferase gene, construct an expression vector (SSRG P::LUC), and use this vector as a reporter vector. The two recombinant vectors were co-transformed into wheat protoplast cells, and the Renilla luciferase overexpression vector (Ubi P::REN) was used as a control. The schematic diagram of the vector is shown in 2A, and LUC/REN represents the SSR gene promoter. expression activity. Fluorescence intensity was measured using the Dual-LuciferaseReporter Assay System kit (Promega, Madison, USA) and a Gloma 20/20 Luminometer (Promega, Madison, USA). As a result, as shown in Figure 2B, TubZIP2 significantly increased the promoter activities of SSRG genes AGPL-cyto , AGPS-cyto and SBEIIa .

(2) Endosperm overexpression of bZIP2 : pJIT163-UBI-hGFP vector (ampicillin resistance) (provided by Gao Caixia, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) is driven by the ubiquitin promoter to drive the expression of GFP. TubZIP2 , TabZIP-A2 , TabZIP-B2 and TabZIP-D2 were recombined into the pJIT163-UBI-hGFP vector, so that the ubiquitin promoter in it drives the massive expression of bZIP2 . The schematic diagrams of the vectors are shown in Figures 2C and 6C, respectively.

The grains of common wheat 15 days after Chinese spring flowering were sterilized with 70% alcohol, and the endosperm was bombarded with a gene gun, so that the above-mentioned recombinant plasmids were transiently expressed in the endosperm, and subsequently cultured in a hypertonic medium in the dark for 48 hours; the total RNA of the endosperm was extracted. , reverse transcribed into cDNA; RT-PCR verified the transient expression of TubZIP2 , TabZIP-A2 , TabZIP-B2 and TabZIP-D2 and their effect on SSRG gene expression, the results are shown in Figure 2D ( TubZIP2 ), and the introduction of empty vector Compared with the wild-type endosperm of the plasmid, TubZIP2 can significantly increase the promoter activity of AGPL-cyto , AGPS-cyto and SBEIIa ; the results of TabZIP2 are shown in Figure 6D, TabZIP-A2 , TabZIP-B2 and TabZIP-D2 can up-regulate SSRG In terms of gene expression, TabZIP-B2 had the greatest up-regulation intensity, and TabZIP-D2 had no obvious effect.

RNA-seq and phenotypic identification of wheat TubZIP2 overexpression lines (1) Screening of wheat transgenic plants: A vector was constructed using the Glu-1Bx14 promoter to drive the overexpression of TubZIP2 , so that the transcription factor was specifically expressed in the grain endosperm, and was studied in the Chinese Academy of Sciences. The transgenic platform of the Institute of Developmental Biology was used for biolistic transformation, and the obtained transformed seedlings were planted in the greenhouse, and their genomic DNA was extracted in small quantities according to conventional methods. SEQ ID No. 6), amplified by conventional PCR, with wild-type wheat genomic DNA as a control, to further detect positive plants. Transgenic wheat homozygous _selfed to the T generation was used for subsequent experiments.

(2) RNA-seq of wheat overexpression line: The endosperm of 7 days, 14 days and 21 days after flowering was taken, and the material was quickly frozen in liquid nitrogen. Duplicate samples were subjected to transcriptome sequencing (RNA-seq). At the same time, RNA was reverse transcribed into cDNA, and Ta4045 (ubiquinol-cytochrome C reductaseiron-sulfur subunit) was used as a control, and the specific primers of SSRG gene were used to verify the expression of SSRG gene by RT-PCR. The results are shown in Figure 3A and Figure 3B. Both RNA-seq and RT-PCR validation results showed that the expression of TubZIP2 was significantly up-regulated in all three stages; Figure 3C and Figure 3D showed that TubZIP2 had no obvious regulatory effect at 7 days after flowering; Figure 3E and 3F, TubZIP2 can significantly increase the expression of AGPL-cyto , AGPS-cyto and SBEIIa genes at 14 days after flowering; Figure 3G and Figure 3H show that the regulatory effect of this gene is weakened at 21 days after flowering, and the SBEIIa gene is mainly up-regulated expression.

(3) Determination of total starch content in mature seeds: The total starch content was determined by a total starch kit (K-TSTA; Megazyme). The results are shown in Fig. 4A, the total starch content of the overexpression lines was significantly increased in Beijing, Hebei Dishang and Zhaoxian compared with the wild type, and the three lines OE82, OE85 and OE87 increased by about 2.88%, OE87 and OE87, respectively. 4.93% and 4.38%. AGPS-cyto and AGPL-cyto are rate-limiting enzymes in starch biosynthesis pathway, and the increase of total starch content was associated with the increase of transcription level of both transgenic lines.

(4) Electron microscope scanning of mature seeds: Cross-section of the mature grains of wild-type and overexpression line wheat was carried out with a blade, and electron microscope scanning of the cross-section was carried out. As shown in Figure 4B, the overexpressed lines had more A-type starch granules, consistent with an increase in total starch content.

(5) Determination of amylose content of mature seeds: The amylose content of the overexpression line and wild-type flour was measured by the DPCZ-II amylose analyzer, and the difference between the total starch content and the amylose content was Amylopectin content. As shown in Figure 4C, the amylopectin content of the overexpression lines at the three sites was significantly higher than that of the wild type, with an average increase of 4.79% in OE82, 8.06% and 7.50% in OE85 and OE87, respectively. This result is consistent with the SBEIIa -catalyzed synthesis of amylopectin. Starch synthesis is consistent. There was no difference in amylose content between Hebei Dishang and Zhaoxian (9.57% reduction in OE87 in Zhaoxian), but the amylose content of OE82, OE85 and OE87 in Beijing decreased by 7.15%, 5.02% and 5.60%, respectively, Fig. 4D is shown. The amylose/amylopectin values of the three overexpression lines at the three sites were significantly decreased due to the increase in amylopectin content and the stable or even decrease of amylose content (amylose/amylopectin in OE82 in Dishang and Zhaoxian County). amylopectin did not change significantly), as shown in Figure 4E.

(6) RVA characteristics of mature seeds of overexpression lines: Rapid viscosity analyzer (RVA) is widely used to evaluate the gelatinization characteristics of starch, the peak viscosity produced by RVA is negatively correlated with the ratio of amylose to amylopectin, and Consistent with the decrease in the straight-to-branch ratio, as shown in Figure 4F, the peak viscosity of wheat in the overexpression lines grown in Beijing all increased significantly. .

(7) Plant phenotype identification of overexpression line wheat: The traits of transgenic wheat in three locations were investigated, as shown in Figures 5A, 5B and 5F for wild-type and overexpression line plants, ear and grain traits, respectively; Figure 5C , 5D and 5E indicate that the plant height, effective tillering and number of spikelets per plant of the overexpression lines are not significantly different from those of the wild type, respectively; Figures 5G, 5H and 5I indicate the grain length, grain width and thousand-grain weight, respectively, where the grain length There were no significant differences, and the grain width and thousand grain weight of the overexpression lines were higher than those of the wild type. Among them, OE85 had the largest increase in grain width, up to 6.62%, and OE82 had the smallest increase, only 3.72%. The increase of kernel width caused the change of 1000-kernel weight. The average 1000-kernel weight of OE85 increased the most, up to 13.50%, and the average increase of OE82 and OE87 was 6.98% and 11.46%.

Phenotypic identification of a wheat TabZIP2 knockout line

(1) Determination of total starch content in mature seeds of knockout lines: The T ₂ generation lines of the TabZIP2 knockout line of wheat planted in Beijing, Hebei Dishang and Zhaoxian from 2017 to 2018 were used for subsequent determination. Total starch content was determined by total starch kit (K-TSTA; Megazyme). As shown in Figure 7A, the total starch content of the knockout lines at the Beijing, Dishang and Zhaoxian sites were all significantly lower than those of the wild type, with an average reduction of 4.18%, 5.69% and 5.69% for bZIP2 ^kn -ab, bZIP2 ^kn -abd1 and bZIP2 ^kn -abd2 5.75%.

(2) Electron microscope scanning of mature seeds of knockout line: Mature grains of wild-type and knockout line wheat were cross-sectioned with a blade, and the cross-section was scanned by electron microscope. The distribution of A and B starch grains in the grains changed with the decrease of total starch. As shown in Figure 7B, the knockout lines contained fewer A-type starch granules.

(3) Determination of amylose content in mature seeds of knockout line: The amylose content of wild-type and knockout line flour was measured by DPCZ-II amylose analyzer, and the difference between total starch content and amylose content was measured. The value is the amylopectin content. As shown in Figure 7C, the amylopectin content of the knockout lines in the three sites, except for the bZIP2 ^kn -ab on the embankment, did not change significantly, and the three lines in the other three sites showed a decrease. bZIP2 ^kn -ab decreased by an average of 3.42%, and bZIP2 ^kn -abd1 and bZIP2 ^kn -abd2 decreased by 5.57% and 10.10%, respectively. The decrease in total starch content and amylopectin content was consistent with the decreased transcriptional activity of TabZIP2 on TaAGPL-cyto , TaAGPS-cyto and TaSBEIIa in the knockout line. (4) RVA characteristics of mature seeds of knockout line: The viscosity of flour was analyzed by RVA, as shown in Figure 7D, the peak viscosity of knockout line was significantly lower than that of wild type. The drop in the amylopectin content of the knockout line caused a significant decrease in the peak viscosity of the RVA.

(5) Grain traits of mature seeds of knockout lines: As shown in Figure 7E and Figure 7F, the grain width and thousand-grain weight of knockout lines at three sites were significantly lower than those of wild type, among which bZIP2 ^kn -ab, bZIP2 ^kn -abd1 and bZIP2 The average grain width of ^kn -abd2 decreased by 3.79%, 5.40% and 5.78%, and the corresponding 1000-grain weight decreased by 9.19%, 14.78% and 15.30% on average. The transcriptional activities of TaAGPL-cyto , TaAGPS-cyto and TaSBEIIa in the TabZIP2 knockout line decreased, resulting in a decrease in the total starch content and amylopectin content of mature grains, which in turn caused a significant decrease in grain width, which further resulted in a significant reduction in 1000-kernel weight compared to wild type. reduce.

<110> Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

<120> A transcription factor bZIP2 that increases the total starch content and amylopectin content of wheat grains and its application

<160> 6

<210> 1

<211> 1722

<212> CDS

<213> TubZIP2, Triticum, Triticum aestivum L.

<400> 1

ATGGCCATCG CGGAGGCGTC CCCCTTCGCG GACCTGCCCT TCCCCGACGA CCTGCCGGAGTTCCCGCACG GCCCCGTCGG GGACGACGAC GCCTTCGCCC TGGACGGCTT CGATCTCGAC GATCTGGACATCGACTTCGA CTTCGACCTC GACCTCGACC TCCTCCCCAC CGACGACGTG CGGCTCCCGT CGCCGCCGCCGCCGCTCGCC ACGTCCTCCT CCTCGGCCGG GTCGCCGGGC GGGGCAGGGG ACTCCTCCTC CGGCTCCGGTGGCGGAGCGG ACGGCGGCCT GAAGAACGAC GAGTCCTCGG AGACGTCTTC GAGGAGCGCG AGCGCTGGGAGCGACGGCAA GGCTAAGGAG GGGGAGGGTG AGGACGACAA GCGGCGGGCG CGGCTGGTGC GGAACCGGGAGAGCGCGCAC CTGTCGCGGC AGAGGAAGAA GCAGTACGTG GAGGAGCTGG AGGGGAAGGT CAAGGCCATGCAGGCCACCA TCGCCGACCT CTCCACCAGG ATCTCCTGCG TCACTGCTGA GAACGCCGCT CTCAAGCAGCAACTGGCCGG TGCTGGTGGA GCAGGCGTCC CGCCGCCGCT GCCGATGTAC CCGGGATTGT ACCCTTTGCCGCTGCCATGG ATGCACCCGG CTTATGCGAT GGGAGCGCGC GGCTCCCAAG TGCCGCTCAT GCCGATACCTCGGCTGAAAA CCAAGCAGCC TGCGTCGGCT GCCGCAGAGC CACCGGCCAA GAAGTCTAGG AAGACCAAGAAGGTTGCCAG TGTTAGCCTC CTTGGATTGC TGTTCCTGAC GATGCTCTGT GGGTGTTTGG TTCCTGCGGTAAATCGGATG TATGGAGCAG TTGATTCCCG AGAAGGAATT GTGCTTGGTC CATCACAATC ACGTCATGGGAGGGTTCTGG CTGTTGACGG G CCTCGAGAT GGTGTCCCGG AAGGTGTTGA TTCGAAGTTG CCACATAATTCAAGTGAGAC GCTCCCGGCC TTGTTGTATA TTCCAAGGAA TGGGAAGCAT GTCAAGATCA ATGGAAATCTTGTTATCCAG TCTGTTGTTG CGAGTGAGAA AGCTTCTTCG CGCATGTGTC ACTCTGATGG GAAGACTTCATGTAACCAAG GACAAGAAGA TACTAGTTTG GCAATTCCTG GCCATGTTGC TCAGTTGAAT TCTGGAGAAGTCATGGAATC TGCTAAAGCA ATAAAAAATA AACTGATGGC TTTACCTCCT GGAGATGGAA GCATATACAGAGACGATGAT GAATTACTGC CACAATGGTT TAGTGAAGCA ATGTCAGGTC CTATGTTGAG TTCCGGAATGTGCACCGAAG TGTTCCAGTT TGATATATCG CCGACCACCA TTGTTCCTGT CTACTCCAGT GGTATGCACAATGCATCACA TAACTCCACG GAGAACCTCC CCTCCAGTCA GTCCCATAAG GTCAAGAACA GAAGGATTTTACATACCATG GCCGTTCCCC TAAAAGGTTC AACGTCCAAC CACACCGATC ACCTCAAAGC GCACCCCAAGAACGAGAGCT TTGCTGGAAA GAAACCGGCT TCATCGGTGG TGGTCTCTGT CCTGGCTGAC CCTAGAGTGGATGCTGATGG AAGGATCTCT TCGAAGTCAT TGTCGCGTAT ATTTGTTGTA GTCCTCGTTG ACAGTGTAAAGTATGTCACT TACTCTTGCG TCCTGCCGTT CAAAACCCAT AGCCCTCATC TG

<210> 2

<211> 1722

<212> CDS

<213> TabZIP-A2, Triticum, Triticum aestivum L.

<400> 2

ATGGCCATCG CGGAGGCGTC CCCCTTCGCG GACCTGCCCT TCCCCGACGA CCTGCCGGAGTTCCCGCACG GCCCCGTCGG GGACGACGAC GCCTTCGCCC TGGACGGCTT CGATCTCGAC GATCTGGACATCGACTTCGA CTTCGACCTC GACCTCGACC TCCTCCCCAC CGACGACGTG CAGCTCCCGT CGCCGCCGCCGCCGCTCGCC ACGTCCTCCT CCTCGGCCGG GTCGCCGGGC GGGGCAGGGG ACTCCTCCTC CGGCTCCGGTGCCGGAGCGG ACGGCGGCCT GAAGAACGAC GAGTCCTCGG AGACGTCTTC CAGGAGCGCG AGCGCTGGAAGCGACGGCAA GGCTATGAAC GGGGAGGGTG AGGACGACAA GCGGCGGGCG CGGCTGGTGC GGAACAGGGAGAGCGCACAC CTGTCGCGGC AGAGGAAGAA GCAGTACGTG GAGGAGCTGG AGGGGAAGGT CAAGGCCATGCAGGCCACCA TCGCCGACCT CTCCACCAGG ATCTCCTGCG TCACGGCTGA GAACGCCGCT CTCAAGCAGCAACTGGCCGG TGCCGGTGGC GCAGGCGTCC CGCCGCCGCT GCCGATGTAC CCTGGATTGT ACCCTTTGCCGCTGCCATGG ATGCACCCGG CTTATGCGAT GGGAGCGCAC GGCTCCCAAG TGCCGCTCAT GCCGATACCTCGGCTGAAAA CCAAGCAGCC TGCGTCGGCT GCCGCAGAGC CACCGGCCAA GAAGTCTAGG AAGACCAAGAAGGTTGCTAG TGTTAGCCTC CTTGGATTGC TGTTCCTGAT GATGCTCTGT GGGTGTTTGG TTCCTGCGGTAAATCGGATG TATGGAGCAG TTGATTCCCG AGAAGGAATT GTGCTTGGTC CATCACAATC ACGTCATGGGAGGGTTCTGG CTGTTGACGG G CCTCGAGAT GGTGTCTTGG AAGGTGTTGA TTCGAAGTTG CCGCATAATTCAAGTGAGAC GCTCCCGGCG TTGTTGTATA TTCCTAGGAA TGGGAAGCAT GTCAAGATCA ATGGAAATTTGGTTATCCGG TCTGTTGTTG CGAGTGAGAA AGCCTCTTCG CGCATGTGTC ACTCTGATGG GAAGACTTCATGTAACCAAG GACAAGAAGA TACTAGTTTG GCAATTCCTG GCCATGTTGC TCAGTTGAAT TCTGGAGAAGTCATGGAATC TGCTAAAGCA ATAAAAAATA AACTGATGGC TTTACCTCCT GGAGATGGAA GCATATACAGAGACGATGAT GAATTACTGC CACAATGGTT TAGTGAAGCA ATGTCAGGTC CTATGTTGAG CTCCGGAATGTGCACCGAAG TGTTCCAGTT TGATATATCG CCGACCACCA TTGTTCCTGT CTACTCCAGT GGTATGCACAACGCATCACA TAACTCCACG GAGAACCTCA CCTCCAGTCA GTCCGATAAG GTCAAGAACA GAAGGATTTTACATTCCATG GCCGTTCCCC TAAAAGGTTC AACGTCCAAC CACACCGATC ACCTCAAAGC GCACCCCAAGAACGAGAGCT TTGCTGGAAA CAAACCGGCT TCATCAGTGG TGGTCTCTGT CCTGGCTGAC CCGAGAGTGGACGCTGATGG AAGGATCTCT TCGAAGTCAT TGTCGCGTAT ATTTGTTGTA GTCCTTGTTG ACAGTGTAAAGTATGTCACT TACTCTTGCG TCCTGCCGTT CAAAACCCAT AGCCCTCATC TG

<210> 3

<211> 1722

<212> CDS

<213> TabZIP-B2, Triticum, Triticum aestivum L.

<400>3

ATGGCCCTCG CGGAGGCGTC CCCCTTCGCG GACCTGCCCT TCCCCGACGA CCTGCCGGAGTTCCCGCACG GCCCCGTCGG GGACGACGAC GCCTTCGCCC TGGACGGCTT CGATCTCGAC GATCTGGACATCGACTTCGA CTTCGACCTC GACCTCGACC TCCTCCCCAC CGACGACGTG CAGCTCCCGT CGCCCCCACCGCCGCTCGCC ACGTCCTCGT CCTCGGCCGG GTCGCCGGGC GGGGCAGGGG ACTCCTCCTC CGGCTCAGGTGGCGGAGCGG ACGGCGCCCT GAAGAACGAC GAGTCCTCGG AGACGTCTTC CAGGAGCGCG AGCGCTGGGAGCGACGGCAA GGCTAAGGAC GGGGAGGGTG AGGACGACAA GCGGCGGGCG CGGCTGGTGC GGAACCGGGAGAGCGCGCAT CTGTCGCGGC AGAGGAAGAA GCAGTACCTC GAGGAGCTGG AGGGGAAGGT CAAAGCCATGCAGGCCACCA TCGCCGACCT CTCCACCAGG ATCTCCTGCG TCACTGCCGA GAACGCTGCT CTCAAGCAGCAGCTGGCTGG CGCCGGTGGC GCAGGCGTCC CCCCGCCGCT GCCGATGTAC CCCGGATTGT ACCCTTTGCCACCGCCATGG ATGCACCCTG CTTATGCGAT GGGAGCGCGC GGCTCCCAAG TGCCGCTCAT GCCCATACCTCGGCTGAAAA CCAAGCAGCC TGCGTCGGCT GCCGCAGAGC CACCGGCCAA GAAGTCTAGG AAGACCAAGAAGGTTGCCAG TGTTAGCCTC CTTGGATTGC TGTTCCTGAT GATGCTCTGC GGGTGTTTGG TTCCTGCAGTAAATCGGATG TATGGACCAG TTGATTCCCG AGAAGGAATT GTGCTTGGTC CTTCACAATC ACGTCATGGGAGGGTTCTGG CTGTTGATGG G CCTCGAGAT GGTGTCTCGG AAGGTGTTGA TTCGAAGTTG CCACATAATTCAAGTGAGAC GCTCCCGGCA TTGTTGTATA TTCCGAGGAA TGGGAAACAT GTCAAGATCA ATGGTAATCTTGTTATCCAG TCTGTTGTTG CGAGTGAGAA AGCTTCTTCA CGCATGTGTC ACTCTGATGG GAAGACTTCATGTAACCAAG GACAAGAAGA TACTAGTTTG GCAATTCCTG GCCACGTTGC TCAGTTGAAT TCTGGAGAAGTCATGGAATC TGCTAAAGCA ATAAAAAATA AACTGATGGC TTTACCTCCT GGAGATGGAA GCATATACAGAGAGGATGAT GAATTACTGC CACAATGGTT TAGTGAAGCA ATGTCAGGTC CTATGTTGAG CTCTGGAATGTGCACCGAAG TGTTCCAGTT TGATATATCG CCAACCACCA TTGTTCCTGT CTACTCCAGT GGTATGCACAACGCATCACA TAACTCCACG GAGAACCTCC CCTCCAGTCA GTCCCATAAG GTCAAGAACA GAAGGATTTTACATTCCATG GCCATTCCCC TAAAAGGTTC AACGTCCAAC CACACCGATC ACCTCAAAGC GCACCCCAAGAACGAGAGCT TTGCTGGAAA CAAACCGGCT TCATCGGTGG TGGTCTCTGT CCTGGCTGAC CCTAGAGTGGATGCTGATGG AAGGATCTCT TCGAAGTCAT TGTCGCGTAT ATTTGTTGTA GTCCTTGTTG ACAGTGTAAAGTATGTCACT TACTCTTGCG TCCTGCCGTT CAAAACCCAT AGCCCTCATC TG

<210> 4

<211> 1722

<212> CDS

<213> TabZIP-D2, Triticum, Triticum aestivum L.

<400> 4

ATGGCCATCG CGGAGGCGTC CCCCTTCGCG GACCTGCCCT TCCCCGACGA CCTGCCGGAGTTCCCGCACG GCCCCGTCGG GGACGACGAC GCGTTCGCCC TGGACGGCTT CGATCTCGAC GATCTGGACATCGACTTCGA CTTCGACCTC GACCTCGACC TCCTCCCCAC CGACGACGTG CAGCTCCCGT CGCCGCCGCCGCCGCTCGCC ACGTCCTCGT CCTCGGCCGG GTCGCCGGGC GGGGCAGGGG ACTCCTCCTC CGGCTCCGGTGGCGGAGCGG ACGGCGGCCT GAAGAACGAC GAGTCCTCGG AGACGTCTTC CAGGAGCGCG AGCGCTGGGAGCGACGACAA GGCTAGGGAT GGGGAGGGTG AGGACGCCAA GCGGCGCGCG CGGCTGGTGC GGAACAGGGAGAGCGCGCAC CTGTCGCGGC AGAGGAAGAA GCAGTACGTG GAGGAGCTGG AGGGGAAGGT CAAAGCCATGCAGGCCACCA TCGCCGATCT GTCCACCAGG ATCTCCTGTG TCACCGCCGA GAACGCTGCT CTCAAGCAGCAACTGGCTGG CGCAGGTGGT GCAGGGGTCC CGCCGCCGCT TCCTATGTAC CCAGGATTGT ACCCTTTGCCACCGCCATGG ATGCACCCGG CTTATGCGAT GGGAGCGCGC GGCTCCCAAG TGCCGCTCAT GCCGATACCTCGGCTGAAAA CCAAGCAGCC TGCGTCGGCT GCCGCAGAGC CACCGGCCAA GAAGTCCAGG AAGACCAAGAAGGTTGCGAG TGTCAGCCTC CTTGGATTGT TGTTACTGAT GATGCTCTGC GGGTGTTTGG TTCCTGCGGTAAATCGGATG TATGGAGCAG TTGATACTCG AGAAGGAATT GTGCTTGGTC CATCACAATC ACGTCATGGGAGGGTTCTGG CTGTTGATGG G CCTCGAGAT GGTGTCTCGG AAGGTGTTGA TTCGAAGCTG CCACATAATTCAAGTGAAAA GCTCCCGGCG TTGTTGTATA TTCCAAGGAA TGGGAAGCAT GTCAAGATCA ATGGAAATCTTGTTATCCAG TCTGTTGTTG CGAGTGAGGA AGCTTCTTCG CGCATGTGTC ACTCTGATGG GAAGACTTCATGTAACCAAG GGCAAGAAGA TACTAGTTTG GCAATTCCTG GCCATGTTGC TCAGTTGAAT TCTGGAGAAGTCATGGAATC TGCCAAAGCA ATAAAAAACA AACTGATGGC TTTACCTCCT GGAGATGGAA GCATATACAGAGACGATGAT GAATTACTGC CACAATGGTT TAGTGAAGCA ATGTCAGGTC CTATGTTGAG CTCCGGAATGTGCACCGAAG TGTTCCAGTT CGATATATCA CCGACCACCA TTGTTCCTGT CTACTCCAGT GGTATGCACAACGCATCACA TAACTCCACG GAGAACCTCC CCTCCAGTCA GTCCCATAAG GTCAAGAACA GAAGGATTTTACATTCCATG GCCATTCCCC TAAAAAGTTC AACGTCCAAC CACACCGATA ACCTCAAAGC GCACCCCAAGAACGAGAGCT TTGCTGGAAA CAAACCGGCT TCATCGGTGG TGGTCTCTGT CCTGGCTGAC CCTAGAGTGGATGCTGATGG AAGGATCTCT TCGAAGTCAT TGTCGCGTAT ATTTGTTGTG GTCCTTGTTG ACAGTGTAAAGTATGTCACT TACTCTTGCG TCCTGCCGTT CAAAACCCAT AGCCCTCATC TG

<210> 5

<211> 20

<212> DNA

<213> Synthetic sequences

<400> 5

ATGGCAGACCACCTTCAAGT

<210> 6

<211> 22

<212> DNA

<213> Synthetic sequences

<400> 6

TCAGTACTTCCACATGCCATCC

Claims (10)

1. the transcription factor that improves grain starch synthesis in Urartu wheat, it is characterized in that: described transcription factor is TubZIP2 gene, or three copies of TubZIP2 gene homologous gene TabZIP2 in common wheat: TabZIP-A2 , TabZIP-B2 and TabZIP-D2 , the TubZIP2 gene, TabZIP-A2 gene, TabZIP-B2 gene, TabZIP-D2 gene, the coding regions of which are respectively composed of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.3 The composition of the nucleotide sequence shown in ID No.4. 2. A recombinant vector comprising the transcription factor of claim 1. 3. An expression vector containing the transcription factor according to claim 1, preferably a plant expression vector. 4. The application of the transcription factor of claim 1 in improving the expression of SSRG gene at grain filling stage in wheat. 5 . The application according to claim 4 , wherein the transcription factor is transformed into wheat, and a positive transgenic line that overexpresses the transcription factor is screened. 6 . 6. the method for overexpressing the transcription factor as claimed in claim 1 in endosperm, it is characterized in that: the endosperm of common wheat is transformed with the overexpression vector containing described transcription factor, preferably, adopt the method of gene gun to transform common wheat flower Endosperm after 15 days. 7 . The primer pair for amplifying the gene of claim 1 , wherein the sequence of the forward primer of the primer pair is shown in SEQ ID No. 5, and the sequence of the reverse primer is shown in SEQ ID No. 6. 8 . 8. the application of the transcription factor described in claim 1 to improve SSRG gene expression and promote starch accumulation in wheat, it is characterized in that: the overexpression vector of described transcription factor is transformed into common wheat, and screening obtains the positive that starch content increases Transgenic lines. 9. The use according to claim 8, wherein the transcription factor is TubZIP2 . 10. the application that transcription factor according to claim 1 reduces SSRG gene expression and starch accumulation in common wheat, it is characterized in that: knock out three copies of gene TabZIP2 with CRISPR/Cas9 technology: TabZIP-A2 , TabZIP-B2 or TabZIP-D2 , and screened out positive transgenic lines.

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