Application of histone deacetylase gene oshdt701 in prolonging plant seed life
A technology of deacetylase and histone, applied in the fields of application, plant products, genetic engineering, etc., to achieve the effect of prolonging the life of plant seeds and great application prospects
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Embodiment 1
[0028] Example 1 Obtaining of overexpressed transgenic rice with histone deacetylase gene OsHDT701
[0029] In order to construct the OsHDT701 overexpression vector, the cDNA fragment of OsHDT701 (accession number: AK072845) was first amplified, and then the fragment was inserted into the vector pUbiO1301 (plasmid map as shown in figure 1 shown), the vector was modified based on pCAMIBA1301, and a maize ubiquitin promoter was added. The constructed vector was transformed into Agrobacterium strain EHA105, and then into the embryogenic callus of mature seeds of rice "Zhonghua 11". Specifically include the following steps:
[0030] 1. Construction of histone deacetylase gene OsHDT701 overexpression vector
[0031] Design specific primers according to the full-length sequence of the histone deacetylase gene 0sHDT701 known by NCBI, and at the same time add BamH I restriction sites to the 5' ends of the primers, and the amplification primers are:
[0032] oxHDT701F: 5'-CG GGATC...
Embodiment 2
[0055] Example 2 Obtaining of Histone Deacetylase Gene OsHDT701 RNAi Transgenic Rice
[0056] The OsHDT701 RNAi transgenic rice material is based on the cDNA sequence of OsHDT701 and its protein conserved domain, using the Zhonghua 11 seedling cDNA as a template to amplify the RNAi target fragment of about 300bp in the OsHDT701 gene, construct it on the interference vector, and use Agrobacterium Rice RNAi transgenic lines were obtained by bacterial dipping technique, and RT-PCR analysis showed that the expression of OsHDT701 gene in the transgenic lines was lower than that in the wild type. Specifically include the following steps:
[0057] 1. Construction of OsHDT701 RNAi vector
[0058] Selecting an appropriate gene segment is the key to successfully constructing an RNA interference vector. The purpose of constructing the vector is to specifically and effectively minimize the expression level of the target gene. Put the cDNA sequence of the target gene on NCBI for comparis...
Embodiment 3
[0084] Example 3 Vitality detection of OsHDT701 overexpression / RNAi transgenic rice
[0085] The determination of seed life is measured according to the classical method, that is, the germination rate after artificial aging treatment (Chen2012, Bentsink et al., 2000; Tesnier et al., 2002; Oge et al., 2008; Rajjou and Debeaujon, 2008). The OsHDT701 overexpression transgenic rice obtained in Example 1 and the OsHDT701 RNAi transgenic rice obtained in Example 2 were respectively subjected to the following experiments:
[0086] 1. Artificial aging treatment
[0087] According to the method of Liu Jun (2000, Journal of Experimental Botany) and Chen (2012, Journal of Experimental Botany), with slight modifications, the artificial aging was completed in a sealed desiccator. Put the seeds in a large centrifuge tube with the cover removed and pretreat them at 15°C and 85% relative humidity (R.H) for 3 days, then transfer them to a well-balanced desiccator at 42°C and 85% R.H for 3-5 d...
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