Screening,Cloning And Functional Identification Of POR Gene In The Albino Of Ananas Comosus Var.BLeaves Using VIGS Technique

Ananas comosus var.bracteatus has become an important new ornamental plant for its diversity and colorful leaves.In order to improve the stability of chimeric characters during the tissue culture and asexual reproduction,it is important to study on the formation mechanism of the chimeric character.In this study,the complete white shoots(CWh)and complete green shoots(CGr)were devived by in vitro tissue culture.Based on the analysis of the differences of physiological indexes and gene expression between the CWh and CGr,the rate-limiting steps and the key genes of chlorophyll biosynthesis in albino leaves were determined.The POR gene(POR)was cloned and its function were verified by VIGS technique.The main results obtained are as follows:1.Ten candidate genes(EF1,UBQ,ACT,GADPH,Histone,TUA,TUB,18 S,elf-5A,α-tubulin)were used for the screening of reference genes for RT-q PCR along the growth of the leaves and between the CGr and CWh leaves.It was confirmed that Histone and α-tubulin were the most suitable internal reference genes along the growth stages.The best combination of 18 s,EF1 and α-tubulin was found in the contrast analysis of CGr and CWh leaves.The expression analysis results of Pet F gene proved that the selected internal reference gene was suitable.2.The content of Pchlide in CGr is 4 times to that of the CWh leaves.The content of Chla in CGr leaves is 30 times to that of CWh leaves.Based on transcriptome sequencing results,it was suggested that inhibition of POR enzyme encoded by POR gene may be a key factor in the formation of the albino cells.3.Two c DNA sequences of POR gene were cloned and named as POR1 and POR2 respectively.The ORF sequences of the two genes were 1167 bp and 1218 bp,encoding 388 and 405 amino acids.The two genes were confirmed to be POR genes by Blast comparison with NCBI.It belongs to the c-like SDR family.The homology of these two nucleotide sequences was 73.81%,and the homology of amino acid sequence was 74.08%.The amino acid sequences were compared with the different POR genes of 39 different plants and the phylogenetic tree was established.It was found that POR1 might be the PORB and POR2 might be the PORA.4.PDS gene was selected as indicator gene and the PDS-TRV2 vector was constructed based on the tobacco rattle virus.The plasmid of PDS-TRV2 and TRV2 were transformed to Ananas comosus var.bracteatus by Agrobacterium(GV3101).PCR detection confirmed that the virus was duplicated and transferred in plants.It was found that the color of test group(Si and Si-Ge)are more yellow than negative control group(CK)and positive control group(P).The content of carotenoids in the Si and Si-Ge groups were lower than that in two control groups,which indicated that the VIGS system was successfully constructed.5.Plasmid of POR1-TRV2 and POR2-TRV2 were transformed to Ananas comosus var.bracteatus by Agrobacterium(GV3101).RT-q PCR results showed that the expression level of POR in test groups(Ti)are reduced significantly compared to two control groups.It was found that the color of test group(Ti1 and Ti1-Ge)are overall yellow,the content of chlorophyll in the Ti1 and Ti1-Ge groups were decreased by30% compared with that of two control groups,it showed that chlorophyll biosynthesis in Ti1 and Ti1-Ge groups was blocked.The leaf color of Ti2 and Ti2-Ge groups are similar to that of the two control groups,and the chlorophyll content of Ti2 and Ti2-Ge groups is not quite different from that of two control groups,it showed that the chlorophyll biosynthesis in Ti2 and Ti2-Ge groups was not affected significantly.It is speculated that POR1 is the key gene in chlorophyll biosynthesis in leaves.