Chlorophyll Synthesis Rate-limiting Gene Mining Of The Albino Cells Of Ananas Bracteatus

Cutting seedlings of chimera and complete white or green plants derived from in vitro culture were used as typical materials in this study. In order to find the differences between albino cells and green cells in genomic DNA and mRNA transcript levels, DNA-AFLP marker analysis, transcriptome sequencing analysis and detection of the content of chlorophyll synthesis precursor material were used to mining the key rate-limiting genes in chlorophyll synthesis. Real-Time PCR was used to detect the expression patterns of genes in chlorophyll synthesis to further validate its possible functions.1. A set of DNA-AFLP analysis technique system was developed in this study, including DNA extraction, silver staining and autoradiography. And it was used to detect the variation of DNA sequence length between the complete white and complete green seedlings.69 pairs of primers were used for AFLP amplification, but no polymorphic band was found between the complete white and complete green seedling. These results indicate that 69 pairs of primers we used did not find significant differences in the genomic DNA length between the complete white and complete green seedling of Ananas bracteatus.2. The leaves, stems, roots and leaves with red color were used for transcriptome sequencing by the Illumina paired-end sequencing technology. A total of 23.5 million high quality sequencing reads and 4.76 Gb bases were obtained. The GC content,Q20 and Q30 were 51,92%,100% and 81%. There are 1,555,808 contigs and 41,052 unigenes the average length of 877.62bp by Trinity stitching software. In total 41,052 unigenes of Ananas bracteatus.,23,275 unigenes were annotated in the NCBI non-redundant protein database and 23,134 unigenes were annotated in the Swiss-Port database. Out of these,17,748 and 8.505 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. Functional annotation against Kyoto Encyclopedia of Genes and Genomes Pathway database identified 5,825 unigenes which were mapped to 117 pathways, and total 39 unigenes involved in the porphyrin and chlorophyll metabolic pathways(Ko:Ko00860).3.The chlorophyll biosynthesis pathway of the albino part of the leaves is hindered. The contents of Chla, Chlb and total Chl of the green part of the leaves are 35.2,16.7 and times to the albino part of the leaves seperately. There is no significant difference in ALA and PBG contents between albino and green leaves. However, there is a significant reduction in the content of UroⅢ,which the green leaf were 4.7 times more than white left.Meanwhile, the contents of CoprogenⅢ, ProtoIX, Mg-ProtoIX and Pchlide in albino levels were significantly low than that of green leaves. This result showed that the transition of PBG to Uro Ⅲ is the rate-limiting step of the chlorophyll biosynthesis of the albino leaves.4.5 pairs of candidate reference genes which can amplify clear band without primer dimers and non-specific amplification were used for qRT-PCR between the leaves of complete white and complete green seedlings. The expression stability of the 5 genes in two samples was validated by geNorm and NormFinder softwares. The results revealed thatl8S rRNA was the most stable reference gene for qPCR analysis of Ananas bracteatus leaf.5.The expression patterns of 12 genes(hemA> hemB> hemC、hemD、hemE、hemG、hemH、 CAO、ACC、ChlD、Chill、ChlM) in chlorophyll biosynthesis pathway between the complete white and complete green seedlings were detected by qRT-PCR. The results showed that the expression levels of 7 genes (hemA、hemC、hemE、CAO、ACC、ChID) in leaves of complete white seedling are reduced compare to the leaves of complete green seedling. The protein of hemC gene catalyze the transition of PBG to UroⅢ,it may play a key role in the albinism of the albino cells.it is suggested that hemC is the rate-limiting gene in the chlorophyll biosynthesis of albino cells of Ananas bracteatus leaves.