Abstract:

【Objective】Construction and normalization of the latex full-length cDNA library would facilitate the identification and analysis of the laticifer-specially expressed genes so as to gain insights into the molecular and regulatory mechanisms of the latex metabolism and the rubber biosynthesis that occur in the laticifers of rubber tree.【Method】Firstly, the primary full-length cDNA library was constructed with the latex of H. brasiliensis (clone Reyan 7-33-97) using recombination of the full-length latex cDNAs with the Gateway donor vector of pDONR222. Secondly, the plasmids of the primary cDNA library were normalized through self-subtraction with the Hevea genome DNA, and the normalized latex full-length cDNA library was constructed using the subtracted plasmids. 【Result】The titer of the unamplified primary cDNA library was about 6.0×106 cfu/mL. the average size of inserted cDNAs was 1.5 kb with a recombination of about 100%,and the ratio of full-length cDNAs was around 76%. The normalized latex full-length cDNA library had a total CFU of 1.87×105 with a recombination ratio of 95%. Quantity RT-PCR results demonstrated that normalization produced about 1 000 fold average reduction of two high abundant latex genes, i.e., small rubber particle protein (SRPP) and rubber elongation factor (REF). 【Conclusion】A high-quality latex normalized full-length cDNA library was successfully constructed, and 12 000 of random clones in this cDNA library were then sequenced. A total of 11 951 ESTs (expressed sequence tag) were attained, and could be assembled into 3 394 unigenes, including 2 061 contigs and 1 333 singlets. The latex cDNA library can be well used for Hevea’s functional genomics, novel gene screening, high-through EST sequencing and the preparation of the latex cDNA microarray of H. brasiliensis.

Key words: Hevea brasiliensis, latex, normalization, full-length cDNA library