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Expression Pattern and Antibody Preperation of Somatic Embryogenesis-related Gene LaSERK1of Larix Leptolepis

Author: LiLong
Tutor: HanSuYing
School: Chinese Academy of Forestry
Course: Biochemistry and Molecular Biology
Keywords: Larix leptolepis Somatic embryogenesis LaSERK1 Subcellular Localization Prokaryotic expression
CLC: S791.22
Type: Master's thesis
Year: 2013
Downloads: 30
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Abstract


Larch, as an important species for fast-growing and afforestation, its technique of somaticembryogenesis has made a breakthrough as a new breeding technology in recent years.However, but there are still some bottlenecks including difficulty of embryonic cells induction,abnormal embryo formation and synchronization difficulty which need to surmount. It is themolecular mechanism research which could bring new change to somatic embryo development.The somatic embryogenesis receptor kinase1（SERK1） gene is considered as an essentialelement involved in the early stage of somatic embryogenesis. In our study we cloned thehomolog of gene SERK1of Larix leptolepis, then we focused on the expression pattern of thehomolog of SERK1of Larix leptolepis in different culture conditions. At last we carried on theprokaryotic expression of LaSERK1extracellular peptides, and utlize it as the antigen for anantibody preparation. Main research results are as follows:(1) We cloned the LaSERK1gene, and translated it into protein, then we did the primarystructure and advanced structure alignments of SERK1genes from some other species andfound that both the sequence and structure of LaSERK1showed a high similarity with thehomologs in other species;(2) qRT-PCR showed that LaSERK1presented a high expression in the earlyembryogenesis cultured in the maturation medium culture which contains hormone ABA, onthe first day it began to rise sharply, on the second day it raised to the highest level, and on thethird day it was also very high, but on the seventh day it decreased obviously, after that itbasically showed a trend of decline. These results suggest that and LaSERK1might perform asignificant role during early somatic embryogenesis of Larix leptolepis, and might be apotential marker of early embryogenic cells;(3) When the embryonic cells are cultured on the simple hormone-free subculture medium,LaSERK1expressed at a very low level, much lower than the cells cultured on the subculture medium contained2,4-D and BA, it suggests that2,4-D and BA has a certain inducing effect toLaSERK1gene expression;(4) We constructed a LaSERK1subcellular localization vector by cloning LaSERK1intothe pSuper1300-GFP vector for the study of subcellular localization of LaSERK1, and provedthe LaSERK1proteins mostly locate on the cell membrane;(5) At last we carried on the prokaryotic expression of LaSERK1extracellular peptides bycloning LaSERK1into the pET28a(+)-His vector, after induced expression, we utlized it as theantigen for an antibody preparation and used ELISA for detecting the antibody titer, theantibody titer we finally got was1:2500. This antibody can be used in the future for thedetection of LaSERK1protein expression at different stages in the process of somaticembrygenesis by performing Western Blot assay, or performing the immunohistochemicalstudy to determine the expression site of LaSERK1protein at different stages in the process ofsomatic embrygenesis.These results suggest that and LaSERK1might perform a significant role during earlysomatic embryogenesis of Larix leptolepis, it is regulated by plant hormones like ABA and2,4-D and it might be a potential marker of early embryogenic cells, LaSERK1proteins mostlylocate on the cell membrane, it may play an important role in responding to the environmentand hormone for the cell signalling transduction.

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CLC: > Agricultural Sciences > Forestry > Forest tree species > Coniferous > Larch
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