Application of histone deacetylase gene to regulation and control on development of rice seed starch

Abstract

The invention belongs to the field of plant transgenes and relates to application of a histone deacetylase OsSRT1 gene to regulation and control on development of rice seed starch. H3K9 acetylation of an OsSRT1 inhibited plant is found to be obviously enhanced, which indicates that the gene is histone H3K9 deacetylase gene. According to research on the transgenic plant which inhibits the gene, the fertility of the plant is obviously reduced, and most of seeds stop developing three days after fertilization and are withered gradually. According to observation, the endosperm starch content of the seeds is reduced two days after the seeds are fertilized. According to Realtime-PCR detection on the change of starch synthesis genes, in the seeds which are fertilized for one day and two day, the expression quantity of the starch synthesis genes of the OsSRT1 inhibited plant is obviously reduced compared with that of the wild type. The OsSRT1 is confirmed to regulate and control starch synthesis by influencing histone acetylation.

Description

Technical field

[0001] The invention belongs to the technical field of plant genetic engineering. Specifically, it relates to the functional verification and application of the histone deacetylase gene OsSRT1 related to the regulation of rice seed starch development. Background technique

[0002] Rice occupies a very important position in my country's food production. The endosperm accounts for more than 90% of polished rice and is the part of rice that is directly eaten. The development of endosperm directly affects the yield and quality of rice. Therefore, the research on genes related to rice endosperm development is of great significance in actual production.

[0003] Current studies have found that the main sites of rice starch synthesis are amyloplasts and chloroplasts, and photosynthesis provides raw materials for their synthesis. The biosynthesis of starch in rice grains is a very complex biochemical process. The products after leaf photosynthesis are first transported ...

Examples

Embodiment 1

[0035] Example 1: Amplification of the full-length cDNA of OsSRT1 gene

[0036] For the gene OsSRT1 (gene accession number LOC_Os04g20270) required by the present invention, the RT-PCR method is mainly used (see: J. Sambrook, EF Fridge, T Maniatis, Huang Peitang, Wang Jiaxi, etc. Translated, molecular cloning The experimental guide (third edition), Beijing, Science Press, 2002 edition) was amplified to obtain the full-length sequence of the OsSRT1 gene. The specific operations are as follows:

[0037] 1) Extract RNA from seedling leaves of rice variety Huanghui 63. The reagent for RNA extraction is Trizol extraction kit from Invitrogen (see the instructions for the kit for specific steps);

[0038] 2) The steps of reverse transcription to synthesize the first strand of cDNA in RT-PCR are as follows:

[0039] ①Prepare mixture 1: total RNA 4μg, DNaseI 2U, 10×DNaseI buffer 1μl, add DEPC (diethyl pyrocarbonate, a strong inhibitor of RNase) treated water (0.01% DEPC) to 10μl, mix well and...

Embodiment 2

[0051] Example 2: Construction of OsSRT1 double-strand suppression vector

[0052] For the genes required by the present invention, through the RT-PCR method (see: J. Sambrook, EF Fridge, T Manniatis, Huang Peitang, Wang Jiaxi and other translations, molecular cloning experiment guide (third edition), Science Press, 2002) Amplify to obtain a specific sequence of OsSRT1. The specific steps are: use the full-length cDNA of OsSRT1 amplified by yourself as a template, search for OsSRT1 specific sequence, design primers, and amplify RNAi inhibitory fragments by PCR. The amplified product was connected to pGEMT-vector (purchased from Promega (Beijing) Biotechnology Co., Ltd., Promega, USA) through T / A cloning for sequencing verification.

[0053] The primers used to clone the RNAi suppression fragment of OsSRT1 are as follows:

[0054] ds-F:5’-GGGACTAGTGGTACCAGTCCTGCAAGAGTTGCAAC-3’

[0055] ds-R:5’-GGGGAGCTCGGATCCCCAGCTTTCACATGCACTAG-3’

[0056] Specific steps are as follows:

[0057] 1) The...

Embodiment 3

[0061] Example 3: Transformation of binary Ti plasmid vector and detection of positive and expression levels of transgenic plants

[0062] 1) The newly constructed suppression expression vector pDS1301-OsSRT1-2 (from Example 2) was introduced into Agrobacterium EHA105 (purchased) by electrotransformation method (references and voltage parameters used are as described in step 2 of Example 2) Among the strains from CAMBIA Laboratory in Australia, the strain that transformed pDS1301-OsSRT1-2 was named pDS1301-OsSRT1-2-EHA105.

[0063] 2) Transform the pDS1301-OsSRT1-2-EHA105 obtained in the previous step into the rice variety Minghui 63. The transformation method refers to the method reported by Hiei et al. (Hiei et al., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J, 1994, 6:271-282.). The T0 generation transgenic plants obtained were named Rn, where n=1,2,3...represented different transgenic...

Technical field

[0001] The invention belongs to the technical field of plant genetic engineering. Specifically, it relates to the functional verification and application of the histone deacetylase gene OsSRT1 related to the regulation of rice seed starch development. Background technique

[0002] Rice occupies a very important position in my country's food production. The endosperm accounts for more than 90% of polished rice and is the part of rice that is directly eaten. The development of endosperm directly affects the yield and quality of rice. Therefore, the research on genes related to rice endosperm development is of great significance in actual production.

[0003] Current studies have found that the main sites of rice starch synthesis are amyloplasts and chloroplasts, and photosynthesis provides raw materials for their synthesis. The biosynthesis of starch in rice grains is a very complex biochemical process. The products after leaf photosynthesis are first transported ...

Examples

Embodiment 1

[0035] Example 1: Amplification of the full-length cDNA of OsSRT1 gene

[0036] For the gene OsSRT1 (gene accession number LOC_Os04g20270) required by the present invention, the RT-PCR method is mainly used (see: J. Sambrook, EF Fridge, T Maniatis, Huang Peitang, Wang Jiaxi, etc. Translated, molecular cloning The experimental guide (third edition), Beijing, Science Press, 2002 edition) was amplified to obtain the full-length sequence of the OsSRT1 gene. The specific operations are as follows:

[0037] 1) Extract RNA from seedling leaves of rice variety Huanghui 63. The reagent for RNA extraction is Trizol extraction kit from Invitrogen (see the instructions for the kit for specific steps);

[0038] 2) The steps of reverse transcription to synthesize the first strand of cDNA in RT-PCR are as follows:

[0039] ①Prepare mixture 1: total RNA 4μg, DNaseI 2U, 10×DNaseI buffer 1μl, add DEPC (diethyl pyrocarbonate, a strong inhibitor of RNase) treated water (0.01% DEPC) to 10μl, mix well and...

Embodiment 2

[0051] Example 2: Construction of OsSRT1 double-strand suppression vector

[0052] For the genes required by the present invention, through the RT-PCR method (see: J. Sambrook, EF Fridge, T Manniatis, Huang Peitang, Wang Jiaxi and other translations, molecular cloning experiment guide (third edition), Science Press, 2002) Amplify to obtain a specific sequence of OsSRT1. The specific steps are: use the full-length cDNA of OsSRT1 amplified by yourself as a template, search for OsSRT1 specific sequence, design primers, and amplify RNAi inhibitory fragments by PCR. The amplified product was connected to pGEMT-vector (purchased from Promega (Beijing) Biotechnology Co., Ltd., Promega, USA) through T / A cloning for sequencing verification.

[0053] The primers used to clone the RNAi suppression fragment of OsSRT1 are as follows:

[0054] ds-F:5’-GGGACTAGTGGTACCAGTCCTGCAAGAGTTGCAAC-3’

[0055] ds-R:5’-GGGGAGCTCGGATCCCCAGCTTTCACATGCACTAG-3’

[0056] Specific steps are as follows:

[0057] 1) The...

Embodiment 3

[0061] Example 3: Transformation of binary Ti plasmid vector and detection of positive and expression levels of transgenic plants

[0062] 1) The newly constructed suppression expression vector pDS1301-OsSRT1-2 (from Example 2) was introduced into Agrobacterium EHA105 (purchased) by electrotransformation method (references and voltage parameters used are as described in step 2 of Example 2) Among the strains from CAMBIA Laboratory in Australia, the strain that transformed pDS1301-OsSRT1-2 was named pDS1301-OsSRT1-2-EHA105.

[0063] 2) Transform the pDS1301-OsSRT1-2-EHA105 obtained in the previous step into the rice variety Minghui 63. The transformation method refers to the method reported by Hiei et al. (Hiei et al., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J, 1994, 6:271-282.). The T0 generation transgenic plants obtained were named Rn, where n=1,2,3...represented different transgenic...

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