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Primary Study On The Function Of Subunit C Genes Of Vacuolar H~+-ATPase In Arabidopsis Thaliana By RNA Interference

Posted on:2009-05-05Degree:MasterType:Thesis
Country:ChinaCandidate:R Q GuoFull Text:PDF
GTID:2120360245465895Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
The plant vacuolar H~+-ATPase(V-ATPase)is a membrane-bound transporter protein,it uses the energy released during cleavage of theγ-phosphate of cytosolic ATP,thereby creating an electrochemical H~+-gradient which is the driving force for a variety of transport events of metabolites.Under stress conditions,the plant can adjust activity of the V-ATPase and become more adaptive to environment.Hence,the V-ATPase was named the stress-responding enzyme.V-ATPase of Arabidopsis contains 13 distinct subunits,and subunit c is the most sensitive component when plants are subjected to stress conditions.In this dissertation,primary function of some VHA-c genes was analyzed by RNAi and the results showed:1.Totally 7 RNAi constructs targeting different regions of the 5 VHA-c genes were made and were transferred into wild-type Arabidopsis,and homozygous transgenic plants from 4 out of the 5 genes were obtained.2.The target gene expression level in the homozygous transgenic plants was detected by semi-quantitative RT-PCR.All the three VHA-c genes checked showed different level silencing,indicating that the RNAi constructions are effective.For example,we obtained 7 VHA-cl transgenic homozygote and 2 lines showed strong decrease in VHA-cl transcript;VHA-c4 have 10 transgenic homozygotes and 4 lines were silenced dramatically;Totally 18 VHA-c5 transgenic homozygote lines were obtained and 5 lines showed medium level silencing.3.The specificity of target gene silencing in the homozygous transgenic plants was determined.The results showed that the construct targeted to VHA-cl is very specific, while both the constructs targeted to VHA-c4 silenced VHA-c2 at the same time,which has a 90.6%identity with VHA-c4 in cDNA sequence.4.Phenotype analysis of transgenic Arabidopsis under normal light condition showed reduction in hypocotyl and root length.Hypocotyl length was reduced by 21%and 29%in cl-4-1 and cl-16-5,respectively,and root length decreased by 9%and 29%in cl-4-1 and c1-16-5,respectively,compared to the control.When the VHA-cl silenced plants were grown under dark condition,the hypocotyl and root length was reduced significantly either.The above results showed that VHA-cl might be responsible for the role of V-ATPase in cell expansion.5.Under salt stress,root length of both VHA-c4 and c2 silenced plants were shorter than the control,indicated that VHA-c4 and c2 genes are critical for the resistance response to high saline stress of V-ATPase.
Keywords/Search Tags:Arabidopsis thaliana, V-ATPase, VHA-c subunit, RNAi, Transgene, Light, Salt stress
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