Cloning,identification And Functional Analysis Of PvLhca1 And PvLhca2 Involved In Color Variation Of Phyllostachys Vivax F.aureocaulis

The Phyllostachys vivax f.aureocaulis is a variant of the Phyllostachys vivax McClure of Bambusoideae.The internodes of the bamboo are yellow,and the different width and depth of green stripes in the internodes of the bamboo stalk are different.Some individuals have whole green or whole yellow culms.The PvLhca1 and PvLhca2 genes in the light systemI(PSI)were identified by SSH subtractive hybridization technique,and the real-time quantitative RT-PCR analysis showed that the expression lelvel of two genes were variant during variation of stalk color bamboo of P.f.aureocaulis.In order to explore the relationship between PvLhca1,PvLhca2 gene and the variation of bamboo stalk color,the full-length sequence of PvLhca1 and PvLhca2 genes were cloned,the expression patterns of PvLhca1 and PvLhca2 genes were analyzed by real-time quantitative RT-PCR in different developmental stages and different degrees of discoloration of internodes,and cDNA sequences were transformed into Arabidopsis thaliana.The main results obtained are as follows:1.The full length of PvLhca1 and PvLhca2 was cloned by RACE technique.The full length of PvLhca1 gene was 741 bp long and encoded protein with 246 aa.The full length of PvLhca2 was789 bp long and encoded a protein with 262 aa.They encoded proteins have three typical transmembrane ?-helices.Phylogenetic tree analysis showed that they were closely identitial with the other plant Lhca gene.2.The four internode of whole green and whole yellow culms were collected,including white internode,pale green internode(pale yellow internode),light green internode(light yellow internode),deep green internode,(deep yellow internode).And three stripy internodes were collected,including pale green stripe,pale yellow stripe,light green stripe,light yellow stripe,dark green stripe,deep yellow stripe.The expression of Pv Lhca1 and PvLhca2 gene in the gradually colored stage of bamboos was decreased analysised by real-time quantitative RT-PCR,which could affecting the attachment of chlorophyll,affecting the stalk color changes.3.Protein was executors of gene function.The antibody was prepared according to PvLhca1 and PvLhca2 protein.The protein abundance of PvLhca1 and PvLhca2 in yellow stripe and green stripeprotein was detected by WB(Western Blot).The results showed that the expression level of PvLhca1 gene in the light green stripe was significantly higher than that in the light yellow stripe,which was consistent with the quantitative RT-PCR results.There was no significant difference in the expression level of protein encoded by Pv Lhca2 gene in light green stripe and the expression level in light yellow stripe.It is suggested that proteins encoded by the PvLhca3 gene could form dimers with the protein encoded by the PvLhca2 gene.4.Construction of plant overexpressing vectors pC1301-PvLhca1 and pC1301-PvLhca2,transformation of A.thaliana,respectively.The results of PCR and RT-PCR showed that PvLhca1 and PvLhca2 were integrated into the genome of A.thaliana and the average number of leaves of transgenic plants was 28 and 25,respectively,which was significantly higher than that of wild type;the ETR(I)(electron transport rate)of transgenic plants is reduced than that of wild-type A.thaliana significantly.These results indicate that overexpression of PvLhca1 and Pv Lhca2 reduce the electron transport efficiency of the light system I,which may lead to changes in the absorption spectrum of the plant.5.The c DNA and DNA sequences of PvLhca1 and PvLhca2 genes derived from the whole green and the whole yellow culm were cloned respectively.The sequence alignment showed that there was no difference in the cDNA of PvLhca1 and PvLhca2 gene from different materials.And the DNA sequences of PvLhca1 and PvLhca2 genes in the whole yellow culm were different from those in the whole green culm.The second intron of PvLhca1 DNA sequence in the whole green culm moved into the third intron of Pv Lhca1 DNA sequence in the whole yellow culm.While the Pv Lhca2 gene DNA sequence in the whole yellow culm was missing all intron sequences compared to the PvLhca2 gene DNA sequence in the green culm.Changes of intron positions and deletions may affect the expression of PvLhca1,PvLhca2 in the whole yellow culm.6.The upstream regulation sequence of PvLhca1 and Pv Lhca2 gene was cloned by gene walk method.The PvLhca1-Green-ATGex is 139 bp,PvLhca1-Yellow-ATGex is 1121 bp,PvLhca2-Green-ATGex is 286 bp,and PvLhca2-Yellow-ATGex is 372 bp.By comparing the sequences of the cloned sequences,the no difference was found in the regulation sequences of the PvLhca1 and PvLhca2 genes between green and yellow clum.The temperature and light responsive element were found in the two sequences by the PlantCARE online software.In conclusion,the cDNA sequences of PvLhca1 and Pv Lhca2 were cloned in this study,transgenic results indicate that overexpression of PvLhca1 and PvLhca2 genes may lead to a decrease in electron transport rate(ETR).Compared with the green culm,the relative expression of two genes decreased at the gradually colored stage of whole yellow culm.WB test also proved that the protein production of the two genes was low,relatively,may lead to which the LHCIcan not be perfectly assembled,then that affects the whole yellow culm chlorophyll deposition and culm color changes.Compared with Pv Lhca1 and PvLhca2 DNA sequences,the changes and deletions of introns could affect the expression patterns of Pv Lhca1 and Pv Lhca2 in yellow culm.