Cloning,Function-verifying And Expression Characterization Of Three Galactinol Synthase(CsGS)Genes In Camellia Sinensis

Raffinose family oligosaccharides(RFOs)is a series of non-reduced sugar,which includes α-galactoside group,and it plays a significant role in risting abiotic stress.Galactinol synthase(GS)is a key gene in the formation of RFOs,while the relative research of GS genes and the relationship between the genes with cold and drought stress and pest feeding of Camellia sinensis is not developed.In this report,we used the whole organs transcriptome gene libraries of shuchazao tea plant(Camellia sinensis)induced by Ectropic oblique attack,then through contrast we find three genes having high homology with other plant GS genes.We cloned three whole length c DNA sequences of CsGS genes based on 5’/3’ RACE techniques mainly described the molecular characterisation,and verified their function by prokaryotic expression,and finally we researched their expression characterization under the stress of low temprature,drought,Ectropic oblique attack and different hormone as well as in different organs of Camellia sinensis through Northern blot and q RT-PCR techniques.1.We obtained three whole length c DNA sequences of CsGS genes based on 5’/3’RACE techniques,named as CsGS1,CsGS2 and CsGS3,whose full length are 1427 bp,1337 bp and 1258 bp,respectively,whose ORF are 1020 bp,1017 bp and 981 bp,respectively,and the number of amino acids were 339,338 and 327,respectively.The three genes have been submitted to the Gen Bank and the registration number was JX624168,KP757767 and KP757768.2.Multiple Sequence Alignment and phylogenetic tree show that CsGS1 and CsGS2 has high homology,which is 92 %.However,both of them have much lower indentity with CsGS3.All of the three CsGSs have three conserved domain,including the dynamic combine site of manganese(DXD),the site of conserved serine phosphorylation(S),and a typical hydrophobicity pentapeptide(APSAA)which is located at the carbon terminal.3.Finding the open reading frame from the three full length c DNA of CsGS genes,and were inserted into PET-32a(+)mobilized into BL21(DE3)strain for inducible expression,the results showed that we can acquire large amount of soluble protein in supernatant induced by 0.25 mg/ml IPTG at 25 ℃ for 3 h and their molecular weight are all about 50 k D.By means of GC/MS,we detect the reaction of the GS enzyme,which show that all of their fusion protein have the specific GS enzyme activity.4.For CsGS1,q RT-PCR and Northern blot results show that after drought treatment for14 d,it up-regulated in some extent.q RT-PCR results show that after low temperature treatment for 3 h,its expression quantity extremely up-regulated,and then rapidly down-regulated to the pretreatment level after 9 h,and it only had little change after the stress treatment of Ectropic oblique attack and different hormone;For CsGS2 and-3,through q RT-PCR,we find that after attacking of Ectropic oblique,they would up-regulate to the max value in 24 h and 6 h,which are 8.3 and 3.3 times than control groups,respectively.After SA treatment,CsGS2 could up-regulate step by step during 9~24 h.After drought treatment,CsGS2 and-3 would up-regulate after 24 h and 9 h,respectively.After Me JA treatment,CsGS3 would extremely obviously up-regulated after 3 h,which is5.3 times than control group and then rapidly down-regulated to the pretreatment level.Other hormone can’t change their expression levels.We find that the expression quantity of CsGS1,-2 and-3 in various kinds of organs are different.Among them,CsGS1 mainly expresses in old leaves and root,and the former expresstion quantity is 750 times of the bud.CsGS2 mainly expresses in old leaves and buds,and the former expresstion quantity is 474 times of the bud.CsGS3 mainly expresses in fruits,and its expresstion quantity is258 times of the bud.Based on the results,CsGS1 may play a vital role in risisting cold stress,and it may play a leading role in resisting stress treatment in leaves.CsGS2 may be related to Ectropic oblique attack,and play a supporting role in resisting stress treatment in leaves.CsGS3 may work on Me JA stress treatment.CsGS3 may make a difference in protecting fruit from various stress or carbon transportation during germination.