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Construction Of Brassica Napus Mutant Library By Activation Tagging And Functional Analysis Of Bn LACS9 Gene

Posted on:2018-03-07Degree:MasterType:Thesis
Country:ChinaCandidate:S ChenFull Text:PDF
GTID:2310330533959362Subject:Biology
Abstract/Summary:PDF Full Text Request
The construction of mutant library plays an important role in the study of functional genomics of Brassica napus.In this study,the activated tag vector pCB260 vector was transformed into Brassica napus by Agrobacterium tumefaciens to construcr T-DNA insertion mutant library.We tested three screening methods to obtain a rapid,efficient and high throughput method.Firstly,we used the Basta to screen T-DNA insert transformants.1000 seeds were seeded in the tray of 28×20 cm,and placed in incubator for about 10-15 days,and then sprayed 5ml / plate 0.01% Basta every other day.The survival plants were transferred to small pots to grow.In these 34 Basta positive plants,33 plants were confirmed with PCR and the rate of transformants was 97%.Secondly,we screened T-DNA insertion plants by green fluorescence.1000 seeds were germinated in ?150mm petri dish with wet filter paper.When seeds have 3mm germ after 12-24 h,GFP signals were detected with Tanon automatic chemiluminescence / fluorescence image analysis system.The 146 GFP positive seeds were transferred to incubator and identified with PCR.101 plants were positive and the rate was 69.18%.Finally,we modified the activation tag vector: replace the green fluorescent protein gene with the red fluorescent protein gene.It has been demonstrated that it was simply to screen out transgenic linseed with a green LED flashlight and a pair of red sunglasses.We cloned the red fluorescent protein gene on the pCX-DR vector and ligated it into the activated tag vector pCB260 by restriction enzyme digestion.The modified tagged vector was transformed into Arabidopsis with Agrobacterium immersion method,but no Arabidopsis seeds with red fluorescence were observed.We subsequently increased the number of transformed Arabidopsis to verify and clone completely expression frame of the red fluorescent protein gene to be express vector.Then,we detected the oil content of the T1 seeds of the transgenic Brassica napus,and oil content of three plants was higher than that of wild-type seeds and two mutants with lower oil content than that of wild type.We also found mutants withdifferent phenotypes such as less branches,more stems,early flowering and infertility in T2 plants.At the same time,we studied the function of the long-chain acyl-CoA synthase 9(BnLACS9)gene.The promoter of the BnLACS9 gene was cloned into the vector pBI121 to drive the GUS reporter gene.The GUS was mainly expressed in the young leaves and flowers in Arabidopsis.In the inhibited plants by the RNAi of BnLACS9 plants,the content of chlorophyll and starch granules of leaves and the oil content of the seeds both decreased.So it can be inferred that the BnLACS9 gene was correlated with rape oil and chlorophyll synthesis.
Keywords/Search Tags:Mutant library, mutant screening, fluorescent protein, BnLACS9 gene
PDF Full Text Request
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