Method for acquiring transgenic plant strain by using celery cabbage intine growth correlated gene PGBc1

A technology of transgenic plants, applied in the field of genetic engineering, can solve problems such as lack of examples

Inactive Publication Date: 2009-08-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, definite evidence is scarce, and examples of application are even more scarce

Method used

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  • Method for acquiring transgenic plant strain by using celery cabbage intine growth correlated gene PGBc1
  • Method for acquiring transgenic plant strain by using celery cabbage intine growth correlated gene PGBc1
  • Method for acquiring transgenic plant strain by using celery cabbage intine growth correlated gene PGBc1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Analysis of spatiotemporal expression characteristics of PGBc1

[0030] Materials used for RT-PCR and Northern hybridization analysis were collected from ajhGMS 'Bcajh97-01A / B'. After the fertility can be distinguished, flower buds of different sizes are taken from fertile lines (divided into 5 grades according to the longitudinal diameter, from grade I to grade V: less than 1mm, 1.2-1.6mm, 1.8-2.2mm, 2.4-2.8mm, 3.0~3.4mm), open flowers, siliques, stem leaves and stems.

[0031] The total RNA of each plant tissue above was extracted and the first strand of cDNA was synthesized for PCR. In order to determine the linearity of RT-PCR, 18, 23, 28, 33 and 38 cycles were compared respectively, and finally it was determined that the number of cycles was 28; the ubiquitously expressed Actin-1 was used as a reference, and the PCR product was within 1.2 % agarose gel electrophoresis and verified by sequencing. The experiment was repeated 2 times.

[0032] Northern h...

Embodiment 2

[0033] Example 2 Using antisense RNA technology to verify the function of PGBc1

[0034] (1) Construction of antisense expression vector

[0035] According to the full-length cDNA sequence, primers were designed between the 232nd base to the 680th base in the PGBc1 sequence, and the 449bp sequence was amplified as an antisense gene fragment, see SEQ ID No.2. And according to the multiple cloning enzyme cutting site of the plant binary vector pBI121, design two PCR amplification primers containing BamH I and Xba I restriction endonuclease sites and three protective bases respectively, and the upstream primer is 5'- TAA GGATCC AATGGAGCAGTGGGAAAT-3', the downstream primer is 5'-GCC TCTAGA TGGACGTTGACATTTGT-3' (the underlined part is the enzyme cutting site). The cDNA of flower buds of 'Bcajh97-01B' was used as a template for PCR amplification. The product was cloned into pGEM-T Easy vector, and the positive cloned plasmid was named pTPGBc1A.

[0036] (2) Construction of Ca...

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Abstract

The invention discloses a method for acquiring a transgenic plant by growing related gene PGBc1 using cabbage pollen intine. The method comprises the following steps: constructing an antisense expression vector; constructing a CaMV35S constitutive promoter expression vector and a BcA9 tissue-specific promoter expression vector; genetically transforming the acquired promoter expression vectors on heart of the cabbage; acquiring PGBc1 gene expressing silent and restricted transgenic plants. The method is helpful for clarifying the whole process of pollen wall growth and molecular mechanism of plant male sterility, and has practical significance to manual controlled plant fertility in production and cultivation of excellent varieties.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular, the invention relates to the application of a gene PGBc1 related to the inner wall development of cabbage pollen. Background technique [0002] The pollen wall is the most characteristic part of the pollen grain. The pollen wall of trinuclear pollen species begins to develop shortly before meiosis, and undergoes a series of complex changes to form a mature pollen wall (Blackmore and Barnes, 1990; Owen and Makaroff, 1995 ). It consists of two layers, the outer pollen exine and the inner pollen inner wall. The main component of the pollen grain exine is sporopollenin, which also contains many proteins, such as those required for participation in self-incompatibility reactions. The inner wall of pollen grains is mainly composed of cellulose, pectin and various proteins. The pollen wall plays a pivotal role in the normal function of pollen, especially in the process of pollen germ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/82A01H1/00C12N15/113
Inventor 黄鹂曹家树张爱红叶意群张豫超
Owner ZHEJIANG UNIV
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