Cloning and application of PcBLFY (Pistacia chinensis Bunge blooming gene)

A technology of Pistacia chinensis and genes, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of Pistacia chinensis with long childhood period, low proportion of female plants, and inability to be applied in production

Inactive Publication Date: 2013-01-16
HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Pistacia chinensis is a dioecious plant. The ratio of male and female plants in natural secondary forests is about 1:1, and about 10% of male plants can ensure sufficient pollen supply. Therefore, there are excess male plants in natural secondary forests, and the female plants that can bear fruit The proportion is low (monoecious individuals are occasionally seen in natural secondary forests, which cannot be used in production at present)
However, Pistacia chinensis has a long childhood period, and it takes 8-12 years to bloom and bear fruit. There is no effective identification method for male and female plants before flowering and fruiting.

Method used

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  • Cloning and application of PcBLFY (Pistacia chinensis Bunge blooming gene)
  • Cloning and application of PcBLFY (Pistacia chinensis Bunge blooming gene)
  • Cloning and application of PcBLFY (Pistacia chinensis Bunge blooming gene)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0196] According to the conserved sequence of cloned model plant LEAFY gene, degenerate primers were designed (forward primer: 5′-GARGTGGCACGTGGSAARAARAA-3′; reverse primer: 5′-CGGAGYTTGGTGGGMACRTACCA

[0197] -3′), using the total RNA of Pistacia chinensis female, male and hermaphroditic plants as templates, RT-PCR amplification was carried out, and a 361bp cDNA sequence was obtained ( figure 1 ), and two gene-specific primers GSP1-1 (primer sequence is 5'-CCTTGAAAACCCTCTCAGTGCATCTGA-3') and NGSP1-2 (primer sequence is 5'-CGCCACGCTCCTTGGCGATGTTCTG-3') for 5'RACE nested amplification , to obtain a 960bp cDNA fragment ( figure 2 ); at the same time, two gene-specific primers, GSP2-3 (primer sequence: 5'-CAGGTCCAGAACATCGCCAAGGAGC-3') and NGSP2-1 (primer sequence: 5'-ATGCCCAACCAAGGTGACAAATCAGG-3') were used to perform 3'RACE nested amplification to obtain 620bp amplified fragment ( image 3 ), the full-length cDNA sequence obtained after splicing was 1570bp.

[0198] Accordin...

Embodiment 2

[0200] Using the difference between the 394th and 603rd base sequences starting from ATG in the coding region (but not limited to these two positions), design the forward primer with the 394th base as the 3' terminal base and the 603rd base The base is the PCR amplification primer of the 3' end base of the reverse primer, which is used to identify the sex of the plant:

[0201] Primer 394F1: 5'-GAGGATTCACGAAGGCGTCACA-3'

[0202] Primer 394F2: 5'-GAGGATTCACGAAGGCGTCACT-3'

[0203] Primer 603R1: 5'-CTGCCTTCAGCATTCTCATCG-3'

[0204] Primer 603R2: 5'-CTGCCTTCAGCATTCTCATCA-3'

[0205] Utilize above two pairs of primers, can carry out sex identification of Pistacia chinensis plant, as following table:

[0206]

[0207] In the table, + indicates that the PCR amplification product can be obtained (for example, a 251bp cDNA amplification fragment can be obtained by RT-PCR), - indicates that the PCR reaction is negative.

Embodiment 3

[0209] Primers were designed (forward primer sequence: 5'-GGAATTCCATATGGATCCTGAAGCATTCA-3'; reverse primer sequence: 5'-CCCAAGCTTGGTCATTCAGTGAGGCTTTAAT-3') to amplify the coding region sequence of PcBLFYF, and inserted into the pET28a prokaryotic expression vector by double enzyme digestion with NdeI / HindⅢ. The constructed recombinant vector was introduced into BL21 strain for expression, and a protein with a size of 43kD was obtained ( Figure 5 ).

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Abstract

The invention discloses a PcBLFY (Pistacia chinensis Bunge blooming gene) and the cloning of the PcBLFY. According to the known LEAFY gene sequence of other plants, a degenerate primer is designed, the total RNA of the male pistacia chinensis bunge, the female pistacia chinensis bunge and the monoecious pistacia chinensis bunge serves as a template, the RT-PCR method is adopted to synthesize a first cDNA chain, and the 5'RACE and 3'RACE methods are adopted to clone the cDNA full-length sequence of the PcBLFY. According to the obtained cDNA sequence, a primer is designed, the RT-PCR method is adopted to obtain the LEAFY genes of which two sequences, one sequence and two sequences having single nucleotide polymorphism difference from the male pistacia chinensis bunge, the female pistacia chinensis bunge and the monoecious pistacia chinensis bunge. The content disclosed by the invention can be used for creating early flowering and fructifying transgenic breeding. The PcBLFY is mainly used for determinating the sex of different pistacia chinensis bunge plantsx and adjusting the sex proportion of the pistacia chinensis bunge energy forest so as to improve the yield dramatically.

Description

technical field [0001] The invention relates to the fields of biotechnology and modern agricultural technology, in particular to the cloning and application of a pistachio flowering control gene (PcBLFY). Background technique [0002] With the depletion of fossil energy, coupled with the environmental impact of toxic gases and greenhouse gases produced during the use of fossil energy, the development of green, renewable and clean energy has become a field of research that countries around the world are vying for. Among them, bioenergy is one of the most promising development directions. my country is a big energy consumer, and 60% of its oil depends on imports. It is of great significance to develop biodiesel to replace limited oil resources. [0003] At present, the bottleneck problem of the biodiesel industry is the supply of raw materials. The operating rate of relevant biodiesel production enterprises is generally low, only about 10%. -70%. Our country has a large pop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/10C12N15/82C12Q1/68A01H5/00
Inventor 吴丽芳张萍萍刘静静郑博李明浩
Owner HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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