CN107151675A - Applications of the acetylase gene OsGCN5 in adjusting and controlling rice drought resisting and root development - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and specifically relates to the application of acetylase gene OsGCN5 in regulating drought resistance and root development of rice. The invention discloses the function of the broad-spectrum acetylase gene in regulating the drought resistance response of rice. An acetylase gene OsGCN5 was obtained by screening the rice database. Its nucleic acid sequence is shown in SEQ ID NO:1, and its encoded protein sequence is shown in SEQ ID NO:2. The gene was constructed on a protein expression vector to express the full-length protein and an in vitro enzyme activity test was carried out to prove that OsGCN5 is a broad-spectrum acetylase. The inhibited expression material of the gene was obtained by transforming the double-stranded inhibitory vector, and the OsGCN5 gene was inhibited After expression, the drought resistance effect of rice is weakened, the expression of some stress response genes is affected, and the development of crown and root related to stress is also affected. It indicated that the gene played an important role in rice drought resistance and root development.

Description

Application of acetylase gene OsGCN5 in regulation of drought resistance and root development in rice

technical field

The invention belongs to the technical field of plant genetic engineering. It specifically relates to the application of the acetylase gene OsGCN5 in regulating drought resistance and root development of rice. The acetyltransferase gene OsGCN5 belongs to the rice GNAT subfamily protein gene. The applicant identified the enzymatic activity characteristics of the gene through in vitro enzyme activity methods, and used the transgenic method to determine the function of the gene in rice drought resistance (including the response to rice drought resistance) Regulation and regulation of root development associated with drought) were analyzed and their potential applications in agricultural production were proposed.

Background technique

Epigenetics is a discipline that gradually emerged at the end of the last century. It is gradually developed from the study of life phenomena that do not conform to the classic Mendelian genetics. Epigenetics refers to heritable variation independent of DNA changes (Bonasio et al., Selective recognition of methylated lysine 9 on histone H3 by the HPl chromo domain. Nature, 2001, 410:120-124). Its material basis is the chromosome space structure formed by DNA winding around the nucleosome octamer. The nucleosome octamer is composed of two copies of histones H3, H4, H2A and H2B, among which the N of the H3 and H4 proteins Various modifications such as methylation and acetylation can be carried out on the terminal lysine residues (Bhaumik et al., Covalent modifications of histones during development and disease pathogenesis, 2007. Nat Struct Mol Biol, 14(11): 1008-1016 ), lysine acetylation modification can relax the nucleosome’s winding effect on DNA, which is conducive to promoting the binding of transcription factors and promoting transcription (Zupkovitz et al., Negative and positive regulation of gene expression by mouse histone deacetylase 1.Mol Cell Biol, 2006, 26(21): 7913-7928. Verdone et al., Role of histone acetylation in the control of gene expression. Biochem Cell Biol, 2005, 83(3): 344-353). To date, a variety of proteins with acetyltransferase activity have been discovered, including GCN5, P/CAF, ESA1, CBP/p300, and Rtt109. Among them, the GCN5 protein belongs to the GNAT subfamily (GCN5-related N-terminal acetyltransferase subfamily), which is a broad-spectrum acetylase that can acetylate multiple sites such as H3K9, H3K14, and H3K27 (Patrick et al., Expanded Lysine Acetylation Specificity of Gcn5in Native Complexes. The Journal of Biological Chemistry, 1999, 274:5895-5900). In the course of subsequent research, people gradually discovered that histone acetylases can modify not only histones but also non-histones. For example, it was found in yeast that GCN5 can regulate the acetylation level of the transcription factor Ifh1 and then regulate its regulation of gene expression ( Downey et al., Gcn5 and Sirtuins Regulate Acetylation of the Ribosomal Protein Transcription Factor Ifh1. Current Biology, 2013, 23:1638-1648). This indicates that GCN5 has a wider range of acetylation functions than histones. In addition, GCN5 is also involved in the formation of the transcription initiation complex, and its acetylation can relax the winding effect of DNA and nucleosomes at the transcription initiation site, making the RNA Pol II complex (RNA polymerase complex) more stable. It is easy to bind to DNA to promote transcription (Featherstone, Coactivators in transcription initiation: here are your orders. Curr Opin Genet Dev. 2002, 12(2): 149-155). So far, the biological function of the GCN5 gene in Arabidopsis has been extensively studied. The growth of the underground and above-ground parts of the gcn5 mutant plants was weakened, and the floral organs were abnormally developed (Vlachonasios et al., Disruption Mutations of ADA2b and GCN5Transcriptional Adapter Genes Dramatically Affect Arabidopsis Growth, Development, and Gene Expression. Plant Cell, 2003, 15: 626-638). In the same year, the research results of Bertrand et al. (Bertrand et al., Arabidopsis Histone Acetyltransferase AtGCN5 Regulates the Floral Meristem Activity through the WUSCHEL/AGAMOUS Pathway. The Journal of Biological Chemistry, 2003, 278:28246-28251) further indicated that AtUSGCNEL5 could pass WUSCHEL/AGAMOUS Pathway. The AGAMOUS pathway regulates the development of floral organs. AtGCN5 regulates the number of Arabidopsis root stem cells through the PLT pathway, thereby affecting root development (Kornet et al.,Members of the GCN5histone acetyltransferase complex regulate PLETHORA-mediated root stem cell niche maintenance and transit amplifying cell proliferation in Arabidopsis.Plant Cell,2009 , 21(4):1070-1079). AtGCN5 can also interact with HD1, TAF1/HAF2 in the flowering pathway to affect the expression of Arabidopsis light-responsive genes (Benhamed et al., Arabidopsis GCN5, HD1, and TAF1/HAF2Interact to Regulate Histone Acetylation Required for Light-Responsive Gene Expression . Plant Cell, 2006, 18:2893-2903). All these results indicate that GCN5 is involved in the response process of Arabidopsis at various developmental stages and environments, and has a very wide range of biological functions.

Rice (Oryza sativa L.) is one of the main food crops for humans, and its yield is not only related to growth and development but also to the ability to cope with various adversities during development. Adversity is divided into biotic stress and abiotic stress, among which abiotic stress includes such as drought, high temperature, low temperature, salt stress and so on. Drought is one of the main factors threatening rice yield. During the long-term evolutionary process, rice has evolved its own set of drought response mechanisms, including drought signal transduction mediated by signaling molecules, activation of stress-related genes, and synthesis of various functional proteins. The proteins that have been identified to participate in the drought response mainly include: molecular chaperones, osmotic pressure regulatory proteins (Tamura et al., Osmotic stress tolerance of transgenic tobacco expressing a gene encoding a membrane-located receptor-like protein from tobacco plants. Plant Physiol ,2003,131:454-462), ion channels (Ward et al., Calcium-Activated K+Channels and Calcium-Induced Calcium Release by Slow Vacuolar Ion Channels in Guard Cell Vacuoles Implicated in the Control of Stomatic Closure.Plant Cell, 1994, 6:669-683), transporter (Klein et al., Disruption of AtMRP4, a guard cell plasma membrane ABCC-type ABC transporter, leads to deregulation of stomatal opening and increased drought susceptibility. Plant Journal, 2004, 39: 219-236) and antioxidant or cell detoxification proteins (Bartels et al., Targeting detoxification pathways: an efficient approach to obtain plants with multiple stress tolerance. Trends Plant Sci, 2001, 6: 284-286). When rice is subjected to adversity stress, a large amount of reactive oxygen species (reactive oxygen species, ROS) will be produced in the cell, which will have a negative impact on cell function. ROS scavenging proteins play an important role in this process, among which peroxidase (Prx) and Glutathione S-transferase (GSTU) is an important enzyme involved in this process (Rezaei et al., Glutathione S-transferase (GST) family in barley: identification of members, enzyme activity, and gene expression pattern. J Plant Physiol, 2013, 170(14): 1277-1284. Shanker et al., Drought stress responses in crops. Funct Integr Genomics, 2014, 14( 1): 11-22). Most of the responses of these downstream genes are regulated by specific transcription factors. Among them, bZIP is a kind of transcription factor family composed of basic amino acids forming a core region adjacent to a leucine zipper. It participates in the process of ABA-mediated gene expression regulation by recognizing ABA response elements. It has been reported that OsbZIP46 in rice is involved in the drought resistance response of rice, and rice overexpressing this gene exhibits a drought-sensitive phenotype (Tang et al., Constitutive Activation of Transcription Factor OsbZIP46 Improves Drought Tolerance in Rice.Plant Physiology, 2012,158:1755 -1768). At the same time, OsbZIP23 is also involved in the salt and drought resistance of rice (Xiang et al., Characterization of OsbZIP23 as a Key Player of the Basic Leucine Zipper Transcription Factor Family for Conferring Abscisic Acid Sensitivity and Salinity and Drought Tolerance in Rice. Plant Physiology, 2008, 148:1938-1952). ABI is another family of transcription factors induced by ABA and involved in stress response.

As a new type of gene regulation mechanism, epigenetic modification is also involved in the stress response. Arabidopsis has reported that a variety of epigenetic-related enzymes participate in the stress response of plants. ROS1, as a brand-new DNA demethylase, was selected in the experiment of brushing and regulating and activating the RD29A promoter (a promoter of Arabidopsis stress response gene), that is, ROS1 can remove the RD29A gene promoter The above DNA methylation promotes the expression of the downstream gene, and the ros1 mutant shows a sensitivity to hydrogen peroxide (Gong et al., ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase.Cell, 2002, 111(6):803-814). HDA9, as a histone deacetylase, is involved in gene silencing effects in Arabidopsis under salt stress and drought stress (Zheng et al., Histone deacetylase HDA9 negatively regulates salt and drought stress responsiveness in Arabidopsis. J Exp Bot, 2016). The GCN5 complex interacts with the cold-inducible transcription factor CBF1 in Arabidopsis, and according to Vlachonasios et al. (Vlachonasios et al., Disruption Mutations of ADA2b and GCN5Transcriptional Adapter Genes Dramatically Affect Arabidopsis Growth, Development, and Gene Expression. Plant Cell , 2003,15:626-638) showed that the up-regulation of COR gene in gcn5 and ada2b mutants was slower than that of wild type during cold induction. These results indicated that GCN5 may be directly involved in regulating the expression of cold-responsive genes.

As a food crop and rice, which is particularly sensitive to drought, there are few reports on how epigenetic modification, especially acetylation modification, participates in the drought response process. We used the rice database (http://rice.plantbiology.msu.edu/index.shtml) to screen a gene homologous to Arabidopsis AtGCN5, named OsGCN5, and used the information provided in the database to identify the gene in the rice cDNA The full-length sequence of the gene was amplified. The yeast expression vector was constructed to express the full-length protein of the gene. The in vitro enzyme activity showed that the protein is a broad-spectrum acetylase gene. Using the transgenic method, we obtained the inhibited expression material of the gene, and found that the gene inhibited expression The drought resistance ability of rice is reduced, and the water loss rate is increased compared with the wild type. The results of RT-PCR showed that the decline of this gene would affect the expression of some stress-responsive genes. At the same time, after further observation of the morphology of the suppressed expression materials, it was found that the number of crown roots decreased significantly. The above results indicated that OsGCN5 can affect the drought resistance response of rice by regulating the expression of stress-responsive genes and the development of rice roots simultaneously.

Contents of the invention

The purpose of the present invention is to overcome the defects of the prior art, and to provide an application of the acetylase gene OsGCN5 in regulating rice drought resistance and root development, that is, to identify a broad-spectrum acetylase gene involved in rice drought resistance response and root (such as crown root) development. The enzyme gene OsGCN5 was analyzed and its application value in rice production was analyzed. The OsGCN5 gene is a broad-spectrum acetylase gene, which is expressed in various tissues. After the expression of this gene is inhibited, the plants show a phenotype that is sensitive to drought and the number of crown roots (Crown Root, CR) decreases; Molecular Biology The results showed that the gene was involved in regulating the expression of some drought resistance genes.

The present invention is realized through the following technical solutions:

From the rice database (http://rice.plantbiology.msu.edu/index.shtml) to screen rice genes homologous to Arabidopsis AtGCN5 (by using protein sequence homology alignment), we named this gene OsGCN5, Its nucleotide sequence is shown in SEQ ID NO: 1, and the sequence length is 1536bp; the 1-1536 position of the sequence is also its coding region (ie CDS), encoding 511 amino acids in total, and its protein sequence is shown in SEQ ID NO: 2 shown.

A non-conserved region between the two domains of the protein (acetylation domain and Bromodomain domain) was amplified, connected to the vector pDS1301 (Figure 1) that inhibits specific gene expression, and then transformed into rice japonica varieties Zhonghua 11 obtained the suppressed material of transgenic rice, extracted transgenic family mRNA, and reverse transcribed it into cDNA to detect the expression level of OsGCN5 in each family. It was found that the expression level of OsGCN5 was significantly suppressed in 7 transgenic families (Figure 2) . The full-length cDNA of OsGCN5 was obtained by amplifying the cDNA of rice leaves, and cloned into the vector of pGEX6p-1 (Fig. 3). The prokaryotic expression strain of E. coli protein was electrotransformed, and the fusion protein of OsGCN5-GST was induced by IPTG. And use GST beads to purify, obtain the fusion protein of purified GST protein and OsGCN5-GST (Fig. 3). OsGCN5-GST fusion protein was incubated with histone H3 or H4 in an in vitro enzyme activity system, and Western Blot was used to hybridize with various acetylated antibodies. It was found that OsGCN5 can directly acetylate K9, K27, K4, K5 alleles of K14 and H4 (Fig. 4), further proved that OsGCN5 is a broad-spectrum acetylase. At the same time, the RNA of rice tissue materials in different periods was used for reverse transcription, and the tissue specificity of OsGCN5 gene expression was detected by qRT-PCR. The results showed that OsGCN5 was expressed in all tissues ( FIG. 5 ). The obtained OsGCN5RNAi family and the control wild-type Zhonghua 11 were subjected to Xiaohongtong drought treatment (the plants were treated when they were in the same state after one month of growth), and it was found that the OsGCN5RNAi family showed a drought-sensitive phenotype (Figure 6A). The experiment found that the OsGCN5RNAi family lost water faster (Fig. 6B). At the same time, the leaves treated with drought for 7 days were extracted, mRNA was extracted and reverse-transcribed, and the expression of stress-response-related genes was detected by qRT-PCR. 7). Studies have shown that roots play a very important role in plant drought resistance (Janiak et al., Gene expression regulation in roots under drought. 2015, J Exp Bot, 67(4): 1003-1014), so we studied OsGCN5RNAi families The root phenotype of the wild-type Zhonghua 11 material was further observed and counted, and it was found that after the expression of OsGCN5 was suppressed, the number of crown roots and root length of the transgenic plants were significantly reduced (Figure 8). The above results indicated that the OsGCN5 gene may participate in the drought resistance response of rice by affecting the number of rice roots and the expression of stress-related genes at the same time.

The invention gist of the present invention that can be summed up is as follows:

1. The application of the broad-spectrum acetylase gene OsGCN5 in regulating the drought resistance response of rice. The nucleotide sequence of the gene is shown in SEQ ID NO:1.

2. The application method of the OsGCN5 gene is as follows: the acetylase activity in the protein encoded by it positively regulates the expression of downstream genes, and positively regulates the expression of some stress-responsive genes during the drought-resistant stress response process of rice leaves, thereby promoting rice. drought response.

3. The application of the broad-spectrum acetylase gene OsGCN5 in the regulation of rice root development. The nucleotide sequence of the gene is shown in SEQ ID NO: 1. The protein encoded by the gene is a broad-spectrum acetylase. regulation of rice root development.

4. The positive regulation of rice root development includes the application of rice crown root number and root length regulation.

Concrete operation steps of the present invention are as follows:

(1) Search the rice OsGCN5 gene (LOC_Os10g28040) homologous to the Arabidopsis AtGCN5 gene on the Rice Genome website (http://rice.plantbiology.msu.edu/index.shtml). According to the sequence published on the website, primers for related vectors were designed, and rice leaf cDNA was used as a template to amplify its full-length cDNA sequence (from the translation initiation site to 1536 bases downstream). The cDNA sequence published on the website is completely consistent, as shown in SEQ ID NO:1. The primer sequences used for amplification are as follows:

OsGCN5-F:5'-GGGGTACCATGGACGGGCTCGCGGCGCC-3',

OsGCN5-R:5'-CGGGATCCTCAGTTCTTCGTTGAGGCTTGGGC-3'

(2) According to the OsGCN5 gene sequence information, the SMART website (http://smart.embl-heidelberg.de/) was used to analyze its protein sequence (SEQ ID NO: 2), and it was found that there were two conserved structural domains in the protein sequence, They are the acetylation functional domain (amino acid 211 to amino acid 287) and the Bromodomain domain (amino acid 396 to amino acid 505), respectively. The sequence between the two conserved domains is the specific sequence of the gene. Based on this, we design Double-strand suppression primer (dsRNAi primer), and using the full-length cDNA in step 1) as a template, obtain the specific DNA of the double-strand suppression segment by PCR amplification, the amplified sequence is shown in SEQ ID NO: 3, and the amplified The primer sequences used are as follows:

OsGCN5-RNAi-F:5'-GGGACTAGTGGTACCGGACAAAGAAAGATGGCAAGG-3',

OsGCN5-RNAi-R:5'-GGGGAGCTCGGATCCGTTGCCTGTAAGTACTGTAATCAGG-3'

(3) The fragment amplified in step 2) was digested with a restriction endonuclease and ligated to the vector to construct the double-stranded suppression segment DNA into the vector pDS1301 to obtain the transformation vector OsGCN5-pDS1301. Utilize Agrobacterium (EHA105)-mediated transgenic method (Hiei et.al., 1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant Journal.6: 271-282) Introduce the transformation vector OsGCN5-pDS1301 into the rice recipient variety Zhonghua 11 to obtain the transformed plant OsGCN5-RNAi (name the transformed plant OsGCN5-RNAi-N in turn, N=0, 1, 2, 3 ...).

(4) Extract the genomic DNA of each transgenic family and use the PCR method to detect positive plants; then extract the mRNA of the positive plants, reverse-transcribe it into cDNA, and use the fluorescent quantitative PCR method to detect the expression of the OsGCN5 gene in each family to obtain Transgenic plants with suppressed expression of OsGCN5.

(5) Amplify the full-length DNA of OsGCN5 with the full-length cDNA of the OsGCN5 gene involved in step (1) and construct it on the pGEX-6p vector (GE Healthcare Life Science), obtain the OsGCN5-pGEX-6p vector, and electroporate The expression strain BL21 was transformed and expressed OsGCN5-GST fusion protein. GST beads (Glutathione Sepharose 4 Fast Flow, GE Healthcare, 17-5132-01) were used to purify and recover expressed proteins (including GST protein and OsGCN5-GST fusion protein), and SDS-PAGE protein gel was run for detection. The sequences of the amplification primers used to construct the vector are as follows:

OsGCN5-6p-F:5'-GGATCCATGGACGGGCTCGCGGC-3',

OsGCN5-6p-R:5'-GTCGACCTCAGTTCTTCGTTGAGGCTTGG-3'

(6) Use the OsGCN5-GST fusion protein purified and recovered in step (5) and histone H3 or H4 to incubate the reaction (GST protein as a control), and use Western Blot technology to detect various acetylated modifications on the histone after the reaction .

(7) RNA was extracted from various tissue parts of rice, reverse-transcribed into cDNA, and the expression level of OsGCN5 in each tissue was detected by fluorescent quantitative PCR.

(8) The OsGCN5 suppressor families and the wild-type control Zhonghua 11 were planted in small red barrels, and the drought treatment was carried out after one month of growth, and the phenotype was observed. After 7 days of drought treatment, the leaves of the OsGCN5 suppressor family and the wild type were taken, mRNA was extracted, reverse-transcribed into cDNA, and the expression levels of stress response-related genes in the two materials were detected by fluorescent quantitative PCR.

(9) Germinate OsGCN5 suppressor lines and wild-type control Zhonghua 11 on square dishes containing rooting medium, observe the root phenotypes and conduct statistical analysis.

Compared with prior art, the present invention has following advantage:

Rice is one of the main food crops for human beings and plays a very important role in ensuring food security. How to improve rice yield and quality is an important scientific issue. However, the relative lack of fresh water resources often seriously affects the yield of rice. With the continuous growth of my country's population, the shortage of water resources will become the bottleneck of food security at this stage. An important way to solve this problem is to improve the drought resistance of rice (Du Hao, Research on stress resistance functions of genes related to hormone and inositol phosphate metabolism in rice, 2013, doctoral dissertation of Huazhong Agricultural University). There have been many studies on the mechanism of rice drought resistance, but how acetylase-mediated histone acetylation is involved in the drought resistance response of rice is not yet clear. The broad-spectrum acetylase gene OsGCN5 involved in the present invention is found to play a very important role in the stress response of rice. The present invention proves that OsGCN5 is a broad-spectrum acetylase through in vitro enzyme activity experiments (Fig. 3, Fig. 4). Genetic transformation of the gene double-strand suppression RNAi vector to obtain the corresponding transgenic plants. Drought stress treatment found that the drought resistance of rice was significantly weakened after inhibiting the expression of this gene, and quantitative fluorescence detection found that the expression of some drought-resistant genes was affected in transgenic plants (Figure 7), indicating that the GCN5 protein affected the expression of some drought-resistant genes. At the same time, through the observation of germination phenotype, it was found that the number of crown roots of OsGCN5-inhibited materials decreased (Fig. 8). The above results indicated that GCN5 protein may affect the drought resistance response of rice by affecting the number of rice roots and the expression of stress genes at the same time.

For a detailed description of the present invention, refer to the content of "Specific Embodiments".

Description of drawings

Sequence Listing SEQ ID NO: 1 is the nucleotide sequence of the broad-spectrum acetylase gene OsGCN5 cloned in the present invention. The sequence length is 1536bp (including the stop codon TGA at the end), encoding 511 amino acids.

Sequence Listing SEQ ID NO: 2 is the protein sequence of the broad-spectrum acetylase gene OsGCN5.

Sequence Listing SEQ ID NO: 3 is the nucleotide sequence of the double-strand suppression RNAi vector (or called transformation vector OsGCN5-pDS1301) constructed by the present invention. The sequence length is 329bp.

Figure 1 is a schematic diagram of the construction of the transformation vector OsGCN5-RNAi (or called the transformation vector OsGCN5-pDS1301) of the present invention. The upper figure is a schematic diagram of the structure of the suppressed expression vector pDs1301 used in the transgenic plant, and the lower figure is a schematic diagram of the position of the specific fragment that is suppressed in the gene (the position of the suppressed segment: 876-1204bp, marked by a yellow box).

Figure 2: Detection of OsGCN5 expression level in OsGCN5-RNAi positive lines of transgenic plants. The ordinate is the relative expression value relative to Actin (an actin gene with very stable expression in rice, accession number is LOC_Os11g06390).

Figure 3: OsGCN5-GST fusion protein expression. Fig. 3A is a map of the expression vector pGEX-6p used in the present invention. The left side of Fig. 3B is the SDS-PAGE protein gel detection picture of Escherichia coli expressing OsGCN5-GST fusion protein. Explanation of reference numerals: from left to right, the lanes are the E. coli sampling samples after induction of expression for 0 hour, 1 hour, 3 hours, and 5 hours, and the sediment and supernatant samples after ultrasonic treatment, and "*" indicates the position where the fusion protein is expressed. The right side of Fig. 3B is the SDS-PAGE protein gel detection picture of the protein recovered and expressed by GST beads (Glutathione Sepharose 4 Fast Flow, GE Healthcare, 17-5132-01).

Figure 4: OsGCN5 in vitro enzyme activity test results. Various acetylation states of histone H3 and H4 after incubation with OsGCN5-GST fusion protein were detected by Western Blot, and GST protein incubation was used as a control. Experiments were repeated twice.

Figure 5: OsGCN5 gene in roots (10-day germination seedlings), young stems (elongating stems), seedlings (5-day germination), young spikes (0.2cm-1cm), leaves (2-month-old rice mature leaves) mRNA level expression detection results. Explanation of reference numerals: the vertical axis is relative to the expression level of Actin.

Figure 6: OsGCN5-RNAi material and wild-type material drought treatment and drought response gene detection. Fig. 6A is the drought treatment test of the small red bucket, respectively, the phenotypes before treatment and after 14 days of drought treatment for one week of rehydration. Fig. 6B is the statistical result of the investigation of the water loss rate of each material.

Figure 7: Detection of relative expression levels of drought-responsive genes in leaves of OsGCN5-RNAi material and wild-type material after drought treatment for 7 days in OsGCN5-RNAi material and wild-type material.

Figure 8: Morphological differences between OsGCN5-RNAi material and wild-type material in primary root (PR), crown root (CR) and aerial parts. Explanation of reference numerals: the left picture in Fig. 8 is the morphological observation photo, and the right picture in Fig. 8 is the experimental statistical data.

detailed description

Embodiment 1: Cloning of OsGCN5 gene

The clone (accession number LOC_Os10g28040) of the gene OsGCN5 of the present invention is mainly obtained by the method of RT-PCR (reverse transcription product PCR amplification) (method is referring to: J. Sam Brook, EF Fritsch, T Maniartis Author, translated by Huang Peitang, Wang Jiaxi, etc., Molecular Cloning Experiment Guide (Third Edition), Beijing, Science Press, 2002 edition). The specific operation steps are as follows:

(1) Extract mRNA from mature leaves of rice variety "Zhonghua 11" (Institute of Crop Science, Chinese Academy of Agricultural Sciences), and use Invitrogen's Trizol extraction kit after mRNA extraction (see the kit's instruction manual for specific steps);

(2) The steps of RT-PCR reverse transcription to synthesize the first strand of cDNA are as follows: ① Preparation of mixed solution 1: 4 μg of extracted total RNA, 1 μl of DNaseI (2U/μl), 1 μl of 10×DNaseI buffer, plus RNase-free Double-distilled water (0.01% DEPC-treated double-distilled water, DEPC is also known as diethyl pyrocarbonate, a strong inhibitor of RNase) 3 μl, after mixing, place the mixture 1 at 37°C for 15 minutes to remove DNA, ②After 15 minutes, place the mixture 1 in a water bath at 65°C for 10 minutes to inactivate DNAse I, and then place it on ice for 5 minutes. ③Add 1 μl of 500 μg/ml oligo(dT) and 1μl of 10mM/L dNTP and mix thoroughly, ④ Immediately place the mixture 1 in a 65°C water bath for 10 minutes, and then place it on ice for 5 minutes to completely denature the mRNA and allow oligo(dT) to bind to the mRNA 3 'End, ⑤ Prepare mixed solution 2: 4 μl of 5×first strand buffer, 2 μl of 0.1M DTT (mercaptoethanol), RRI, reverse transcriptase SSⅢ 1 μl, mix well and place mixed solution 2 in a water bath at 42°C for 1.5 hours , ⑥ After the reaction, the mixture 2 was denatured in a 90°C dry bath for 10 minutes, ⑦The final reaction product was stored at -20°C, and all reagents used in the reaction were purchased from Invitrogen.

(3) Then design primers according to the full-length cDNA sequence of the OsGCN5 gene provided by the Rice Genome database (http://rice.plantbiology.msu.edu/index.shtml) and use PCR to amplify the full-length. The specific PCR system is: reverse transcription cDNA product 1μl, 10×PCR buffer 2μl, 10mM/L dNTP 2μl, forward primer (F) and reverse primer (R) 0.4μl each, LA-Taq enzyme 0.2μl, double distilled 12 μl of water (the PCR buffer, dNTP, Taq enzyme, etc. used were all purchased from TAKARA Company). The PCR reaction program is as follows: ① 94°C for 5 minutes, ② 94°C for 30 seconds, ③ 56°C for 30 seconds, ④ 72°C for 120 seconds, ⑤ cycle from ② to ④ 30 times, ⑥ 72°C for 7 minutes, ⑦ 4°C storage. The primers used to clone the full length of OsGCN5 are:

OsGCN5-F:5'-GGGGTACCATGGACGGGCTCGCGGCGCC-3',

OsGCN5-R:5'-CGGGATCCTCAGTTCTTCGTTGAGGCTTGGGC-3'

The final amplified OsGCN5 full-length connection - T Easy vector (Promega Company), using T7 and SP6 primers to sequence and find that the amplified nucleotide sequence is completely consistent with the sequence provided by the Rice Genome database, as shown in SEQ ID NO:1. The primer sequences used for sequencing are:

T7:5'-ATTATGCTGAGTGATATCCC-3',

SP6:5'-TAAATCCACTGTGATATCTT-3',

Embodiment 2: Construction of double-strand suppression dsRNAi vector

According to the full-length cDNA sequence of the OsGCN5 gene published in the Rice Genome database (http://rice.plantbiology.msu.edu/index.shtml), the SMART website (http://smart.embl-heidelberg.de/) was used to analyze its The encoded protein sequence (SEQ ID NO: 2) has two conserved domains, namely the acetylation functional domain (amino acid 211 to amino acid 287) and the Bromodomain domain (amino acid 396 to amino acid 505). Design double-strand suppression primers (dsRNAi primers) for the non-conservative region in the middle of the two conserved domains, and use the full length of the gene cloned in Example 1 as a template for PCR amplification to obtain double-strand suppression segment DNA. Spe I and Kpn I restriction endonuclease sites were added to the 5' end, and Sac I and BamH I restriction endonuclease sites were sequentially added to the 5' end of the downstream primer. After the target sequence is amplified by PCR, the amplified product is connected to the -T Easy vector (Promega Company) was sequenced to verify its amplified DNA fragment (SEQ ID NO:3 in the sequence table). The target DNA fragment was digested with this vector and connected to the subsequent vector pDS1301. Specific steps are as follows:

(1) The T/A cloning plasmid with the RNAi fragment of OsGCN5 is digested with Kpn I and BamH I restriction endonucleases, and the target band is recovered, and the carrier plasmid pDS1301 (made by It was transformed by Chu Zhaohui, a graduate of the State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University; the map of the plasmid is shown in Figure 1; see the literature: Chu et al., Promoter mutations of an essential gene for pollen development result in disease resistance in rice.Genes Development , 2006, 20:1250-1255.) to carry out ligation reaction at 16°C for 6 hours (the endonucleases used were all purchased from Bao Biological Engineering Dalian Co., Ltd., and operated according to the product manual provided by the company; the ligase was purchased from Promega Company );

(2) The ligation product was electrotransformed into competent Escherichia coli (DH10B strain), and a single clone was picked for subsequent sequencing verification to obtain a cloning plasmid with the first strand connected to pDS1301.

(3) Use Spe I and Sac I to digest the same cloning plasmid with the RNAi fragment of OsGCN5, and use Spe I and Sac I to digest the vector constructed in 2), and use the above ligation reaction to connect the foreign fragment and the carrier 1. Transform into E. coli competent and verify by sequencing with vector primers. The primers used to construct the double-stranded suppression dsRNAi vector are:

OsGCN5-RNAi-F:5'-GGGACTAGTGGTACCGGACAAAGAAAGATGGCAAGG-3',

OsGCN5-RNAi-R:5'-GGGGAGCTCGGATCCGTTGCCTGTAAGTACTGTAATCAGG-3'

The finally obtained OsGCN5 gene double-strand suppression RNAi vector (or called the expression vector OsGCN5-pDS1301).

Example 3. OsGCN5 enzyme activity verification in vitro

(1) Using the OsGCN5 full-length vector involved in Example 1 as a template to design primers and amplify the full-length OsGCN5 gene, the 5' ends of the left-end primer (OsGCN5-6p-F) and right-end primer (OsGCN5-6p-R) were Add BamH I and Sal I restriction sites to amplify fragment ligation -T Easy vector (Promega Company), the ligation reaction conditions are 16° C., 6 hours. Escherichia coli competent (DH10B strain) was electrotransformed, and single clones were selected for subsequent sequencing to obtain clones without mutations. Use BamH I and Sal I restriction endonucleases to digest the vectors obtained from the above clones respectively to obtain exogenous DNA fragments, and at the same time use BamH I and Sal I restriction endonucleases to digest the OsGCN5-pGEX-6p vector, and carry out the two Ligation reaction, electrotransformation of Escherichia coli competent to select a single clone and sequence to obtain the OsGCN5-pGEX-6p vector (the endonucleases used were purchased from Bao Bioengineering Dalian Co., Ltd., and operated according to the product manual; the ligase was purchased from Promega Company) . The sequences of the amplification primers used to construct the vector are as follows:

OsGCN5-6p-F:5'-GGATCCATGGACGGGCTCGCGGC-3',

OsGCN5-6p-R:5'-GTCGACCTCAGTTCTTCGTTGAGGCTTGG-3'

(2) Import the constructed OsGCN5-pGEX-6p vector into the E. coli expression strain (BL21 strain) by electroporation, select a single clone, and expand the culture. When the strain is shaken to OD value=0.5, add IPTG (iso Propylthiogalactoside, Shanghai Sangong Bioengineering Technology Co., Ltd.) to a final concentration of 0.2mM/L, and samples were taken at 0 hour, 1 hour, 3 hours, and 5 hours. , 200mM/L NaCl, 0.5% NP-40 (Sigma, 18896), 1mM/L EDTApH=8.0, 0.01g/ml Protease Inhibitor (Roche, 04693159001)) resuspended, and after processing with a sonicator (SONIPREP 150, the United States) Centrifuge at 12000rpm for 5 minutes to separate the precipitate and supernatant samples, which are dissolved in protein loading buffer (recipe: 62.5mM Tris-HCl, pH6.8; 2% SDS; 25% glycerol (glycerol); 0.01% bromophenol blue ( Bromophenol Blue); 10% β-mercaptoethanol (β-mercaptoethanol), denatured at 95°C for 10 minutes, and placed on ice for 5 minutes. It was 15% SDS-PAGE protein gel for electrophoresis (electrophoresis using Mini -PROTEAN3 electrophoresis tank system, operate according to the instructions). Verify whether the protein is expressed (Figure 3).

(3) Purify and recover the supernatant expressed in step (2) using GST beads (Glutathione Sepharose 4 Fast Flow, GE Healthcare, 17-5132-01). The specific steps are as follows: wash the GST beads with buffer for two days, and centrifuge at 500g Remove the supernatant, add the supernatant in 2), mix the beads and the supernatant overnight at 4°C, centrifuge at 500g to remove the supernatant, resuspend the beads in buffer, mix well and shake at 4°C for 10 minutes, centrifuge to remove the supernatant, and wash like this 6 times. Remove the supernatant. Add 100 μl GST dissolution buffer (15.366mg-30.732mg GlutathioneReduced (Shanghai Sangong Bioengineering Technology Co., Ltd.), 250 μl 1M/L Tris-HCl pH=8.0), dissolved at 4°C for half an hour. Centrifuge at 500g to get the supernatant, take 15 μl of the supernatant and add protein loading buffer to denature at 95°C for 10 minutes, place it on ice for 5 minutes, and carry out electrophoresis detection on a 15% SDS-PAGE protein gel (electrophoresis adopts Bio-Rad company's Mini-PROTEAN3 electrophoresis tank system, operated according to the instructions of the electrophoresis tank), as shown in Figure 3, the applicant purified relatively clean GST empty protein and OsGCN5-GST fusion protein.

(4) Use the protein purified and recovered in step (3) to incubate with histone H3 or H4, and use Western Blot technology to detect the state of histone acetylation modification (method reference: Tie et al., 2009.CBP-mediated acetylation of histone H3lysine 27 antagonizes Drosophila Polycomb silencing. Development. 136(18):3131-3141). Incubate the reaction as follows: 40mM/LTris-HCl pH=8.0, 0.1M/L NaCl, 10% glycerol, 0.1mM/L EDTA, 1mM/L DTT, 1mM/L PMSF, 5mM/L sodium butyrate, 0.1mM/L acetyl-CoA (Sigma Company), 10 μl histone H3 or H4 (Sigma Company). Incubate at 30°C for 4 hours. The Western Blot technique is as follows: transfer the membrane using the Mini Trans-Blot Cell transfer tank system of Bio-Rad Company, according to its instructions, the histones are transferred to the Hybond-P PVDF membrane of Amersham Company, containing 2% bovine serum albumin ( BSA) in PBS buffer (NaCl 137 mmol/L, KCl 2.7mmol/L, Na2HPO ₄ 4.3mmol/L, KH ₂ PO ₄ 1.4mmol/L, pH 7.5) was blocked at room temperature for 2 hours, with a volume ratio of 1:10000 Add different antibodies (as shown later) and incubate at room temperature for about 1.5 hours. The antibodies used were: H3 (ab1791, Abcom Company), H3K9Ace (07-352, Millipore Company), H3K27Ace (07-360, Millipore Company), H3K4Ace (07-539, Millipore Company), H3K14Ace (04-1044, Millipore Company) company), H4K16Ace (07-329, Millipore Corporation), H4K5Ace (04-118, Millipore Corporation), H4 (ab70701, Abcom Corporation). The primary antibody solution was recovered, and the membrane was washed six times with PBS buffer for 10 minutes each time, and then HRP-labeled goat anti-rabbit secondary antibody or anti-mouse secondary antibody (purchased from SouthernBiotech, Inc., available at Bo Biotechnology Co., Ltd. (Beijing) agent), incubated at room temperature for 1.5 hours, then washed the membrane with PBS buffer six times, 10 minutes each time, and used the SuperSingnal Pico (purchased from Pierce Company) kit to perform X-ray films according to the instructions. Develop and record the picture with a scanner. The results are shown in Figure 4, indicating that OsGCN5 can directly acetylate H3K9, K27, K4, K14 and H4K5 sites, and is a broad-spectrum acetylase.

Example 4. Functional verification of OsGCN5 in regulating rice drought resistance response

1. Transformation of binary Ti plasmid vector and detection of positive transgenic plants and expression levels:

(1) Transform the rice recipient variety "Zhonghua 11" with the transformation vector pDS1301-OsGCN5 obtained in Example 2, and the transformation method is in accordance with the standard method of the State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University (for example: Patent No. ZL 2011100940899, The name of the invention: the application of histone demethylase gene OsJ5 in improving rice resistance; patent publication number: CN 102732535; patent authorization date: patent document on August 28, 2013). The obtained transgenic plants of the T0 generation were named OsGCN5-RNAi (named OsGCN5-RNAi-N in sequence, N=0, 1, 2, 3...).

(2) The leaves of transformed plants of the T0 generation planted in the field were taken and the total DNA was extracted. The DNA extraction method was the CTAB method (Zhang et al., genetic diversity and differentiation of indica an japonica rice detected by RFLP analysis, 1992, Theoretical and Applied Genetics, 83, 495-499). The positive detection of the second-strand primers (pMCG2F and pMCG2R) of the double-strand suppression T0 generation transformed plants was carried out by PCR method. The PCR vector primers used are as follows:

pMCG2F: 5'-GGCTCACCAAACCTTAAACAA-3',

pMCG2R: 5'-CTGAGCTACACATGCTCAGGTT-3'.

(The above primers were provided by Shanghai Sangong Bioengineering Technology Co., Ltd. Synthesis)

The PCR reaction system formula is: template 2 μl (about 100ng), 10×PCR buffer 2 μl, 10mM/L dNTP 2 μl, forward primer (pMCG2F) and reverse primer (pMCG2R) 0.4 μl each, r-Taq enzyme 0.2 μl, supplement 12 μl of double-distilled water (the PCR buffer, dNTP, Taq enzyme, etc. used were all purchased from TAKARA Company). The PCR reaction conditions are as follows: ① 94°C for 4 minutes, ② 94°C for 30 seconds, ③ 56°C for 30 seconds, ④ 72°C for 1 minute, ⑤ cycles from ② to ④ 32 times, ⑥ 72°C for 7 minutes, and ⑦ storage at 4°C. PCR products were detected by electrophoresis on 1% (mass/volume) TBE agarose gel. The fragments amplified by the second-strand primers of the vector (pMCG2F and pMCG2R) are unique to transgenic plants, and the corresponding fragments cannot be amplified in wild-type plants. Harvest seeds from positive plants of the T0 generation (called T1 generation) to prepare for the investigation of plant traits of the T1 generation. (3) In order to detect the expression level of the target gene in the pDS1301-OsGCN5 transgenic plants, the applicant analyzed the expression of the transgenic T0 generation plants by fluorescent quantitative PCR. Extract the total mRNA of the transgenic positive plants of the T0 generation and carry out reverse transcription. The extraction reagent is the Trizol extraction kit of Invitrogen Company (operated according to the kit instructions). Proceed as follows:

① Prepare mixed solution 1: 4 μg of extracted total RNA, 1 μl of DNaseI (2U/μl), 1 μl of 10×DNaseI buffer, add RNase-free double distilled water (double distilled water treated with 0.01% DEPC, DEPC also known as Diethyl pyrocarbonate, a strong inhibitor of RNase) 3 μl, after mixing, place the mixture 1 at 37°C for 15 minutes to remove DNA;

② After 15 minutes, place the mixture 1 in a water bath at 65°C for denaturation for 10 minutes to inactivate DNAse I, and then place it on ice for 5 minutes;

③ Add 1 μl of 500 μg/ml oligo(dT) and 1 μl of 10 mM/L dNTP to the mixture 1 and mix well;

④ Immediately place the mixture 1 in a 65°C water bath for 10 minutes, then place it on ice for 5 minutes to completely denature the mRNA and allow oligo(dT) to bind to the 3' end of the mRNA;

⑤ Prepare mixed solution 2: 4 μl of 5×first strand buffer, 2 μl of 0.1M DTT (mercaptoethanol), RRI, reverse transcriptase Mlv 1 μl, mix well and place mixed solution 2 in a water bath at 42°C for 1.5 hours;

⑥After the reaction, put the mixed solution 2 in a dry bath at 90°C for 10 minutes to denature;

⑦ Store the final reaction product at -20°C, and all reagents used in the reaction were purchased from Invitrogen.

After the product was obtained, the expression level of OsGCN5 was detected by real-time fluorescent quantitative PCR. Reagents were purchased from Treasure Bioengineering Dalian Co., Ltd., and the reaction system was referred to the instruction manual. The PCR instrument is 7500 from ABI Company in the United States. The PCR parameters are 95°C for 10 seconds, denaturation at 95°C for 5 seconds after entering the cycle, annealing and extension at 60°C for 40 seconds, and 45 cycles. The primer sequences used in Real-time PCR are:

OsGCN5Realtime-F: 5'-CAGATAACAATGCCGTTGGCT-3'

OsGCN5Realtime-R: 5'-TGACGCCTAATCATCGTTGC-3'

The results of OsGCN5 expression are shown in Figure 2, and the families with better suppression of OsGCN5 gene expression were 2, 6, 10, 14, 18, 20, and 48.

2. Character investigation and expression analysis of T2 generation transgenic plants:

(1) Expression of OsGCN5 gene in different tissue parts

Different tissue parts of the rice variety Zhonghua 11 were collected, and the mRNA was extracted using the Trizol extraction kit from Invitrogen (see the kit’s instruction manual for specific operation steps). After reverse transcription, the OsGCN5 gene was detected by fluorescent quantitative PCR. The expression levels in roots (germinated 10-day seedlings), young stems (elongating stems), seedlings (germinated for 5 days), young ears (0.2cm-1cm), leaves (2-month-old rice mature leaves), and the results As shown in Figure 5. The figure shows

Different tissues of the rice variety Zhonghua 11 at different growth stages were collected, RNA was extracted with Trizol extraction kit from Invitrogen Company (see the instruction manual of the kit for specific operation steps), and the OsGCN5 gene was detected with a fluorescent quantitative PCR instrument after reverse transcription Expression levels in roots (germinated 10-day seedlings), young stems (elongating stems), seedlings (germinated for 5 days), young ears (0.2cm-1cm), leaves (2-month-old rice mature leaves), results As shown in Figure 5, it can be seen from the figure that the gene is expressed in various tissues and organs, but the expression level is the highest in leaf tissue, which shows that OsGCN5 is a gene constitutively expressed in tissues and organs.

(2) Detection of the role of OsGCN5 gene in rice stress resistance

The OsGCN5 suppressor families and wild-type control Zhonghua 11 that germinated for 10 days were planted in small red barrels (half were planted with OsGCN5 suppressor families; half were planted with wild-type Zhonghua 11). When the two grew to one month, the rice plant size Basically the same as the growth state. Pour out the open water in the bucket and keep it in a state of non-watering to make the soil slowly lose moisture. After 14 days of drought treatment, the plants show a very obvious drought phenotype. The main manifestation is that the leaves curl and slowly wither. At this time, rewatering begins. Let the plants regain water and observe the phenotype (reference to the small red barrel drought test: Tang et al., Constitutive Activation of Transcription Factor OsbZIP46Improves Drought Tolerance in Rice, 2012, Plant Physiology, 155, 1755-1768.), as shown in Figure 6A Show. In order to further confirm the above observed phenotypes, we took the OsGCN5-suppressed family grown for one month under the same conditions and the wild-type Zhonghua 11 as control materials to test the water loss rate of detached leaves. Specific method: Cut off the leaves of one-month paddy plants at the base with scissors, and immediately weigh the fresh weight (G _0h ), then weigh the material weight (G _xh ) every hour, and the water loss rate corresponding to x hours (G _xh -G _0h )/G _0h (method reference: Tang et al., Constitutive Activation of Transcription Factor OsbZIP46 Improves Drought Tolerance in Rice. Plant Physiology, 2012, 158:1755-1768). The statistics of the results are shown in Figure 6B, the water loss efficiency of the OsGCN5-inhibited family was higher than that of the control wild-type Zhonghua 11. At the same time, in order to find the downstream genes that may be affected by OsGCN5, we took the leaves of the OsGCN5 suppressor family and the wild type Zhonghua 11 after 7 days of drought treatment, extracted the mRNA, reverse transcribed it into cDNA, and detected the stress response genes in the two species by fluorescent quantitative PCR. According to the expression level in the materials, some stress-responsive genes such as peroxidase (Prx) and glutathione S-syltransferase (GSTU) are found to be involved in the removal of ROS (reactive oxygen species) under stress and some transcription factors related to stress Genes such as ABI and bZIP-like genes were down-regulated in OsGCN5-suppressed families relative to wild-type. It shows that OsGCN5 participates in regulating the expression of some stress-responsive genes during the drought stress process of rice ( FIG. 7 ). The primer sequences used for detection are as follows:

LOC_Os11g10460-F:5'-CTGGGGTCTCAGAAGATGGAA-3'

LOC_Os11g10460-R:5'-CAGTTTCACTCGGCAACAAATC-3',

LOC_Os02g14430-F:5'-TGTGAGATTACCATGGCTTCGA-3'

LOC_Os02g14430-R:5'-GGAGGAAGAAGGCCAGCAA-3',

LOC_Os03g04240-F:5'-GCGACAAGACTCTTGTTGATTCA-3'

LOC_Os03g04240-R:5'-CCCGTCGTCTGGGAACTG-3',

LOC_Os10g38540-F:5'-GTCACTGCTTTGGGCCTTTG-3'

LOC_Os10g38540-R:5'-TGACCCGGAAGAACATCACA-3',

LOC_Os01g68370-F:5'-GGATGATATTTGATCAGTAACCCGTAGT-3'

LOC_Os01g68370-R:5'-CAACGATGAACAACCAAACCAT-3',

LOC_Os03g19370-F:5'-GCCACAGATCGAGATGCTGTAC-3'

LOC_Os03g19370-F:5'-CGTAGCCCTGCAGCATACTG-3',

(3) Phenotype observation of OsGCN5 gene during rice crown and root development

Since the rice root system is closely related to the drought resistance response of rice, we investigated the development of crown roots in OsGCN5-suppressed families. The details are as follows: germinate 11 flowers in each suppressed family of OsGCN5 and the control wild type on a square dish containing rooting medium, observe the phenotype after 7 days, and make statistics on the height of the aerial part, the length of the underground part and the number of crown roots. It is found that OsGCN5 inhibits The underground part of the family was significantly weakened, especially the number of crown roots was significantly reduced (Fig. 8), indicating that OsGCN5 may participate in the drought response of rice by affecting the development of rice roots.

Claims (4)

1. The application of the acetylase gene OsGCN5 in regulating the drought resistance response of rice, characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO:1. 2. The application according to claim 1, characterized in that, the application method of the OsGCN5 gene is: the acetylase in the protein encoded by it positively regulates the expression of downstream genes, and positively regulates the expression of the rice leaf during the response process of drought resistance and adversity. The expression of some stress-responsive genes promotes the drought-resistance response of rice. 3. The application of the acetylase gene OsGCN5 in the process of regulating rice root development, characterized in that the nucleotide sequence of the gene is as shown in SEQ ID NO: 1, and the protein encoded by the gene is a broad-spectrum acetylase. Positive regulation of rice root development. 4. The application according to claim 3, wherein said positively regulating the development of rice roots includes the number of crown roots and root length of rice.