Cloning And Function Analysis Of RCA Large Isoform And Small Isoform Genes Of Pinus Massoniana

Pinus massoniana is a native species in China and has high economic and environmentalvalue because it grows rapidly and has strong adaptability to the environment and Wide range ofapplications. So it’s important to study the functional gene of P. massoniana, especially itsphotosynthesis-related genes. In the process of plant photosynthesis ribulose-1,5-bisphosphatecarboxylase/oxygenase(RubisCO) is a key enzyme as well as a rate-limiting enzyme, but itsactivity is very low. RubisCO activase(RCA) is the enzyme that required in the activation ofRubisCO in plant. We studied the rca of P. massoniana and got the following results.Two full-length cDNA fragments of RCA genes named PMrca1(accession numberKF420118) and PMrca2(accession number KF420119) were isolated from P. massonianathrough RT-PCR and RACE. Their sequences length are1816and1953bp and open readingframe of them are1443bp and1323bp, encoding480and440amino acids respectively. Thesetwo proteins belong to AAA (ATPase associated with various cellular activities) superfamily andhave the conversed sequences of RCA and chloroplast transit peptide. Besides the affinitybetween the RCA large isoform of P. massoniana and RCA large isoform of Acer rubrum is78%and so is the affinity between small isoform of Pinus massoniana and RCA small isoform of Acerrubrum.To construct PMrca1and PMrca2plant expression vectors, full sequences cDNA of PMrca1and PMrca2were cloned and inserted into pBI121plasmid respectively through restrictionenzyme digestion and ligation. And then the plant expression vectors constructed weretransformed into tobacco by leaf disc method. We got9transgenic plants of PMrca1and7transgenic plants of PMrca2after resistance screening and molecular detection.Observing morphological phenotypes of transgenic tobacco and wild tobacco we found thatcompared with the wild-type tobacco, the leaves number of transgenic tobacco of PMrca2dramatically increased, while the other transgenic tobacco had no significant phenotypic changes,indicating that over express of RCA small isoform of P. massoniana in tobacco is in favor ofdifferentiation and growth of tobacco leaf and RCA large isoforms of P. massoniana do not havemuch influence on tobacco’s morphological phenotypes.By detecting the net rate of photosynthesis on light intensity response curve of wild-typetobacco and transgenic tobaccos, we found that comparing transgenic tobacco of PMrca1andPMrca2with wild-type tobacco, the maximum net photosynthetic rates of the front two wereincreased by36%and23%, respectively, and their the apparent quantum yields were increasedby32%and16.2%too. But both the light saturation points of transgenic tobacco of PMrca1andPMrca2were taken in advance of37.5%, and their the dark respiration rate and lightcompensation point declined dramatically at the same time. All these results indicated that both RCA large and small isoforms of P. massoniana could improve tobacco’s photosynthesis rateand sensitivity to light, while their overexpression could also cause tobacco dark respiration rateincreasing.Both Pinus massoniana RCA large isoforms and small isoforms with green fluorescentprotein fusion protein plant expression vectors were constructed through gateway technology.By inserting open reading frame of PMrca1into the pET-28a plasmid we constructedPMrca1prokaryotic expression vector named pET-rca1. The best concentration of IPTG forinducing the expression of pET-rca1is1.0mM and the suitable inducing time is8h.