Antisense expression method for self-compatibility novel germplasm of S-RNase creation pear by utilizing pollen mediators

An antisense expression, pollen-mediated technology, applied in the field of molecular genetics, can solve the problems of pollen tube inability to elongate, inability to complete pollination and fertilization, inability to set fruit, etc., to avoid tedious and tedious, high efficiency, and avoid tissue culture process Effect

Active Publication Date: 2013-11-06
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0004] The self-incompatibility of pear is controlled by multiple alleles at a single locus, including the S gene that controls the self-incompatibility of style and pollen respectively. When the S genotype of pollen and the S allele in style At the same tim...

Method used

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  • Antisense expression method for self-compatibility novel germplasm of S-RNase creation pear by utilizing pollen mediators
  • Antisense expression method for self-compatibility novel germplasm of S-RNase creation pear by utilizing pollen mediators
  • Antisense expression method for self-compatibility novel germplasm of S-RNase creation pear by utilizing pollen mediators

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Embodiment 1

[0038] 1. Antisense expression vector pGSA1285-S 3 build

[0039] According to the S registered in NCBI 3 -Full-length RNase gene mRNA sequence, designed primer pair S 3 PF: 5'-TGCCTCGCTCTTGAACAAA-3' (SEQ ID NO.1), S 3 PR: 5'-TATATACTAGCTAGCCCCGC-3' (SEQ ID NO. 2). Extract style RNA from ‘Fengshui’ pear, reverse transcribe it into cDNA, use the synthesized cDNA as a template, and use S 3 PR / S 3 PF primers were used for PCR amplification to obtain S 3 - The full length of the RNase gene cDNA sequence. The amplification reaction system is 25 μL, including 2.5 μL 10×Buffer, 2.0 mmol L -1 MgCl 2 , 0.2mmol·L -1 , 0.1ummol·L -1 Forward and reverse primers, 0.2 μL Taq enzyme (Takara Company) and 60 μg cDNA. The PCR amplification program was as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, after 38 cycles, extension at 72°C for 10 min, and 10°C for 10 min. figure 1 As indicated, using the...

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Abstract

The invention discloses an antisense expression method for self-compatibility novel germplasm of an S-RNase creation pear by utilizing pollen mediators and belongs to the molecular genetics field. According to the antisense expression method for the self-compatibility novel germplasm of the S-RNase creation pear by utilizing the pollen mediators, an antisense expression vector pGSA1285-S3 is built through a double-enzyme-digestion method, the antisense expression vector pGSA1285-S3 is transferred to a juicy pear with yellow pear pollen serving as a vector through a method of processing the pollen mediators through ultrasonic waves, three plants containing the antisense expression vector are screened out according to molecular identification of progeny strains, and the novel germplasm of the self-compatibility pear is obtained. The antisense expression method for the self-compatibility novel germplasm of the S-RNase creation pear by utilizing the pollen mediators has the advantages of overcoming a defect that the existing self-compatibility pear is rare in variety resource, providing a new way for cultivating and innovating varieties of the self-compatibility pear, meanwhile providing good testing materials for functional identification of S-RNase gene protein products and having good breeding practical significances and theoretical research values.

Description

technical field [0001] The invention belongs to the field of molecular genetics and relates to a method for creating pear self-compatibility new germplasm by using pollen to mediate antisense expression of S-RNase. Background technique [0002] The pear tree is the third largest fruit tree in my country, and it is also a traditional fruit for foreign exchange earning in my country. It has many varieties and is widely distributed. It is cultivated all over the country except Hainan Province. Pear is a typical gametophytic self-incompatibility fruit tree of Rosaceae. Few of the currently cultivated varieties show self-fertilization. Therefore, pollination trees or artificial pollination must be arranged in production to obtain corresponding yield. From the point of view of species evolution and utilization of heterosis, self-fertilization is beneficial to plants. However, for the fruit tree production for the purpose of harvesting fruit, it is undoubtedly unfavorable that the...

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Application Information

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IPC IPC(8): C12N15/82C12N15/66A01H5/00
Inventor 吴俊张绍铃马倩倩齐开杰黄小三吴巨友
Owner NANJING AGRICULTURAL UNIVERSITY
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