Cloning And Quantitative Analysis Of Genes Related Ascorbate Biosynthesis In Actinidia Eriantha

This paper studies the growth curve and content variation of Ascorbic Acid and AsA related enzyme Gal LDH and APX which in Actinidia eriantha Benth variety ‘Ganmi 6’ during the process of fruit development. Extracted RNA by using improved lithium chloride precipitate method from Actinidia eriantha Benth fruitlets and the full-length cDNA of synthetase genes GalUR, GME, GMP and cDNA sequence of GalUR were cloned by RT-PCR. Expressions of GME, GMP, GalUR, GPP, GalLDH, GalDH and GGP were analyzed by qPCR. The study will be helpful in further analysing physiology and molecule mechanism of AsA synthetase in Actinidia eriantha. The main results are showed as follow:1 As A cotent of all the growth piriod was determined, it accumulated rapidly at fruit development earlier stage, met the maximum(22.43mg/g)at 53 days after full blossom, and then decended to 7.65mg/g at 112 days after full blossom, balanced to mature period at 6.56 mg/g. Activity change trend of Gal LDH kept a high level during fruit development. It hit the maximum(1.95U/g FW) at DAFB53 d, while fall to the minimum when fruit riped. Activity change trend of APX was stronger at later period. Activity of APX reach the maximum(1.957U/g FW) in fructescence.2 It showed homology analysis of these cDNA showed high homology with the cDNA from other plants, respectively. The full-length of GalUR cDNA is 1031 bp and coding for 376 aminoacid with 95% homology with actinidia deliciosa. The full-length of GME cDNA is 1139 bp and coding for 376 aminoacid with 98% homology with actinidia deliciosa and actinidia rufa; The full-length of GMP cDNA is 1392 bp coding for 361 aminoacid, the sequence showed that it was 97% homologous identity to that of GMP from actinidia latifolia.3 Expressions of GME, GMP, GalUR, GPP, GalLDH, GalDH and GGP were analyzed by qPCR. Expressions of GalUR had a similar trend with AsA contend change trend which increased at earlier stage and then dropped during fruit development.Which showed D-galacturonic acid pathway may act on Actinidia eriantha AsA synthetase. GME and GMP rised violently in 34-53 d, then circuitous dropped. Expressions trend of GPP、GalDH、GalLDH were similar, met the maximum at 34 d, then decreased slowly. Change trend of GPP expressions was descenting. The maximum appeared at DAFB53 d, and the minimum appeared at DAFB95 d. Expressions of synthetase genes in L-galactose pathway provide evidence that L-galactose pathway exist in Actinidia eriantha AsA synthetase. Expressions of GME kept a high level during fruit development, which is 3 to 5 times than other genes. Which can be inferred that L-gulonic pathway also act on Actinidia eriantha AsA synthetase.