Application of gene PHYB for controlling rice drought stress toleration

A drought stress, rice technology, applied in the direction of genetic engineering, plant genetic improvement, application, etc., can solve the problems of blindness of drought-resistant molecular breeding, and achieve the effect of improving drought stress tolerance

Inactive Publication Date: 2011-09-07
山东省农业科学院高新技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to the lack of understanding of the molecular mechanism of plant drought resistance, there is still a lot of blindness in molecular breeding for drought resistance

Method used

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  • Application of gene PHYB for controlling rice drought stress toleration
  • Application of gene PHYB for controlling rice drought stress toleration
  • Application of gene PHYB for controlling rice drought stress toleration

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Isolation and cloning of DNA fragments used to construct antisense PHYB gene plant expression vectors

[0023] Total RNA was extracted from leaves of rice variety Nipponbare (a publicly reported variety) using TRIZOL reagent (Invitrogen). The specific steps are as follows: put 20 mg leaves into a liquid nitrogen pre-cooled mortar, add liquid nitrogen and quickly grind them into powder, put the powder into a 1.5ml centrifuge tube, quickly add 1ml Trizol (Invitrogen) and invert to mix well, and leave at room temperature Let stand for 5 minutes. Centrifuge at 12,000 rpm for 10 minutes at 4°C, and transfer the supernatant to a new 1.5ml centrifuge tube. Add 200 μl of chloroform, shake vigorously by hand for 15 seconds, and let stand at room temperature for 2-3 minutes. Centrifuge at 12000 rpm for 15 minutes at 4°C. Take the colorless aqueous phase into a new 1.5ml centrifuge tube, add 250μl isopropanol, 250μl high salt solution, invert and mix well, and let st...

Embodiment 2

[0025] Embodiment 2: Construction and genetic transformation of antisense PHYB gene plant expression vector

[0026] In order to better analyze the function of PHYB, the applicant reduced the expression level of PHYB gene in rice through antisense technology. The function of the gene was studied according to the phenotype and physiological characteristics of the transgenic plants.

[0027] The construction method of the antisense PHYB gene plant expression vector is as follows: first, the positive clone pMD18-PHYB cDNA obtained in Example 1 was double-digested with KpnI and XbaI, and the insert was recovered; Plant expression vectors, recovery of vector fragments. Use the recovered insert and vector fragments for ligation to transform Escherichia coli XL1-Blue. Positive clones were screened by enzyme digestion to obtain a plant expression vector named pIG121Hm-anti-PHYB (see figure 1 ). pIG121Hm-8 is obtained by replacing the SacI site between the coding region and the ter...

Embodiment 3

[0118] Example 3: Detection of PHYB protein levels in transgenic rice plants and wild-type rice

[0119]The rice variety Nipponbare and 5 independent T3 transgenic rice plants were used as materials to extract the soluble protein of rice leaves at the 5-leaf stage, and the level of PHYB protein in rice leaves was detected by western blot. The specific method is as follows; 1 g of rice leaves were collected from the above materials, thoroughly ground in liquid nitrogen, and 2 ml of protein extraction buffer (100 mM Tris-HCl, pH 8.3, 5 mM EDTA, 0.2% β-mercaptoethanol and protease inhibitor mixture) was added , mix well, and place on ice for 30 minutes. Then centrifuge at 12000×g for 15 minutes at 4°C. Transfer the supernatant to another tube, add 2 / 3 volume of saturated ammonium sulfate, mix well, and let stand on ice for 30 minutes. Centrifuge at 12000×g for 30 minutes at 4°C. Discard the supernatant and suspend the pellet with 200 μl protein extraction buffer. Protein conc...

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Abstract

The invention discloses the application of a gene PHYB for controlling rice drought stress toleration, belonging to the technical field of gene engineering. In the invention, a full-length code area of a rice PHYB gene is amplified by a PCR method and is reversely connected with a plant expression vector pIG121Hm-8, the rice is genetically transformed into a rice variety (Oryza sativa L. japonica. cv. Nipponbare), and the endogenous PHYB gene expression of the rice is inhibited to obtain a transgenic plant with inhibited rice PHYB gene expression. In a genetically modified T3 generation plant, the drought stress toleration of a positive rice plant is obviously higher than that of a negative rice plant. Under the same conditions, after the plants are dehydrated and condensed, above 70 percent of genetically modified positive plants can restore normal growth, and only 5-10 percent of corresponding wild plants can restore normal growth.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to the application of a gene PHYB for controlling drought stress tolerance of rice. Background technique [0002] Drought has become a worldwide problem. The world's arid and semi-arid areas have accounted for more than 1 / 3 of the land area. The impact of drought on plants ranks first among many natural adversity factors. Food loss caused by drought accounts for more than half of all natural disaster food losses. The Second National Agricultural Census Leading Group Office of the State Council, the Ministry of Land and Resources and the National Bureau of Statistics issued the Second National Agricultural Census Main Data Bulletin (No. 6) on February 29, 2008. More than half of the cultivated land is dry land. Under natural conditions, drought stress not only seriously affects the growth and yield of crops, but also limits the distribution of plants. Therefore, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/00
Inventor 谢先芝刘婧周晋军钱凤芹毕玉平范仲学
Owner 山东省农业科学院高新技术研究中心
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