Construction Of Antisence And RNAi Expression Vector For α-Farnesene Synthase Gene And Its Genetic Transformation Into 'Fertility' Pear

Superficial scald (scald) is a physiological disorder that affects fruits such as apple and pear, characterized by tissue damage at the surface of the fruit, resulting in fruit quality damage.Many experiments proved thatα-farnesene has been associated with the occurrence of scald. The accumulation ofα-farnesene in the skin of apple fruit during storage appears to be predominantly through classical mevalonate pathway 3-Hydroxy-3-methylglutaryl CoA reductase (HMGR), farnesyl diphosphate synthase (FPPS) andα-farnesene synthase (AFS) is the rate-limiting enzyme in the mevalonate pathway. AFS is the final, rate-limiting enzyme that converts farnesyl diphosphate (FPP) toα-farnesene in the mevalonic acid (MVA) pathway. Getting molecular mechanism of superficial scald throughα-farnesene synthase has become a very heated topic.In this study, specific primers according to the full sequence ofα-farnesene synthase gene were designed, antisence and RNAi plant expression vectors forα-farnesene gene were constructed and transformed into pear to furtherly learn the function ofα-farnesene synthase. Also, the regeneration system of pear was further improved, and factors related to the regeneration of pear were studied. The results were as follows:1. RNA with high quality was isolated from pear cultivars and got the fragments for expression vectors using RT-PCR.2. A double-stranded RNAi plasmid vector was constructed by ligating the 515 bp fragment from PFS cDNA gene to 396 bp fragment in inverted directions, the results were confirmed by restriction enzymes digestion and sequencing that the constructed RNAi plasmid contained the designed structure.3. A recombinant antisence expression vector PBI-PFS-L forα-farnesene was also constructed. The two vectors were successfully transferred to pear through PCR test.4. On the basis of regeneration system from in vitro leaves of pear, factors influencing the frequency of gene transfer were examined and an efficient gene transformation system for pear was established.