Liriodendron chinensis LhPIN3 genes and application thereof
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A technology of hybridizing Liriodendron tulipifera and genes, which is applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of insufficient auxin and achieve good application prospects
Active Publication Date: 2014-08-27
NANJING FORESTRY UNIV
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Problems solved by technology
[0005] The polar transport of auxin is closely related to many growth and development processes of plants, but the regulatory mechanism of auxin is not fully understood in many places
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Embodiment 1
[0028] Using the hybrid Liriodendron tulipifera as material, total RNA was extracted and reversed into cDNA, corresponding primers were designed for PCR, after agarose gel electrophoresis, the target band was recovered, connected with pMD19-T vector, transformed into Escherichia coli, sequenced and analyze. Pick positive clones for plasmid extraction, add restriction sites, and simultaneously double-enzyme cut with the vector pBI121, ligate under the action of T4 ligase, transfer into Agrobacterium GV3101, and pass through the flower organs after Arabidopsis reaches the right age. The soaking transformation method was used for transformation, and the phenotypes of the regenerated plants of T1, T2 and T3 generation transgenic homozygous were observed.
[0029] (1) Extraction of total RNA
[0030] Using the lateral buds of the hybrid tulip tree as material, according to the NORGEN kit ( Norgen Biotek ) for RNA extraction, and the reagents and consumables used are all treated ...
Embodiment 2
[0045] Example 2 Gene function verification
[0046] First build the tulip tree LhPIN3 Expression vector, transformed into Arabidopsis pin1 In mutants, the observation of Liriodendron LhPIN3 Can the mutant restore the phenotype, compare the phenotype difference between the two, speculate that Liriodendron LhPIN3 Function; construction of 35S: PIN3 expression vector, transformed into Arabidopsis wild type, observed phenotype, speculated that Liriodendron chinensis LhPIN3 Function.
[0047] 1) Construction of the carrier
[0048] The Escherichia coli strain used in the present invention is E. coli JM109 (purchased from Biovector (Dalian) Co., Ltd.); the expression vector is pBI 121 (purchased from Biovector Co., LTD).
[0049] The specific process is as follows:
[0050] 1. Through PCR in LhPIN3 XbaI and SmaI restriction sites were added upstream and downstream of the gene, respectively. The PCR system and reaction conditions are the same as the full-length amplificatio...
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Abstract
The invention discloses liriodendron chinensis LhPIN3 genes and application of the hybrid liriodendron chinensis LhPIN3 genes. A nucleotide sequence of the hybrid liriodendron chinensis LhPIN3 genes is shown as SEQ ID NO.1. According to the hybrid liriodendron chinensis LhPIN3 genes, on the basis that a mature hybrid liriodendron chinensis somatic embryogenesis system is established, auxin is cloned to transport the LhPIN3 protein genes; through expression analysis, it is proved that the hybrid liriodendron chinensis LhPIN3 genes have larger effect on embryogenic cell development and somatic embryo growth in the process of hybrid liriodendron chinensis somatic embryogenesis; after overexpression experiments, it is found out that overexpression of the LhPIN3 can influence growth and development of plants; in addition, the expression of the LhPIN3 can save partially pin1 mutants, with functions completely lost, of the LhPIN3 genes of arabidopsis thaliana, can enable the phenotypes of the pin1 mutants to recover and enable the pin1 mutants to bloom and yield legumes; accordingly, the liriodendron chinensis LhPIN3 genes can be applied to somatic embryogenesis of the plants, adventitious root induction, adventitious bud induction and differentiation, and the like and has good application prospects.
Description
technical field [0001] The invention belongs to the technical field of plant genetic engineering, in particular to a hybrid tulip tree LhPIN3 Genes and their applications. Background technique [0002] In the 20th century, the important progress of biotechnology in the discovery and cultivation of fine materials and new varieties of forest trees was the application of somatic embryogenesis technology and regeneration plant cultivation technology. Somatic embryogenesis technology has become an important means of rapid asexual reproduction of fine forest resources due to its characteristics of bipolarity, fast reproduction speed, and high efficiency, and it can be combined with bioreactors to achieve large-scale commercial production. The essence of somatic embryogenesis is the problem of cell differentiation, which makes somatic cells develop into somatic embryos and then develop into a complete plant. The molecular basis of cell differentiation is the result of differentia...
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