Over-Expression Effects Of Rubisco Activase Gene (CsRCA) In Cucumber

Cucumber （Cucumis sativus L.） is an important cold-sensitive vegetable cultivated insolar-greenhouse. Ribulose^-1,5-bisphosphate carboxylase/oxygenase （Rubisco） is a keyenzyme in the photosynthetic carbon reduction cycle, and play an important regulatory rolein the photosynthesis. Although the Rubisco is more in plant leaves, its catalytic efficiency isvery low, the carboxylation and oxygenation activities of internal Rubisco can only beactivated by Rubisco activase （RCA）. Thus, it is promising to elucidate the promoting effectof RCA gene on growth and photosynthesis of cucumber by cloning RCA gene and genetictransformation through genetic engineering.In the present study, the RCA gene （CsRCA） was introduced into inbred line of cucumberwith the agrobacterium-mediated method. The transgenic plants were screened by PCR, andtheir expression in mRNA and protein level were analysed by real-time PCR and western blotrespectively. The over-expression effects of CsRCA were elucidated. The results of the presentresearch were as follows:1. The restriction enzyme result showed that the CsRCA was inserted into the binaryvector pCAMBIA1301, a sense expression vector containing CsRCA gene was constructed.2. A recombinant of prokaryotic expression vector pET-CsRCA was constructed andtransformed to E. coli. BL21（DE3） plysS, and expression was induced with IPTG. The stronginduced fusion protein was used to immunize BALB/c male mice to obtain primary antibody.Blood was collected and the sera was tested as1:100. The results showed that the antibodyobtained was qualified for cucumber RCA protein analysis.3. A high efficient and stable cucumber genetic transformation was constructed. Thecotyledonary node was choosed as the most suitable explant, the optimal culture medium ofshoot induction of cucumber was MS+2.5mg·L^-16-BA+0.5mg·L^-1ABA+2mg·L^-1AgNO₃,and the optimal culture medium of elongation and rooting was MS+0.2mg·L^-1KT+0.5mg·L^-1IAA, the most suitable concentration of Hygromycin used as resistant selection was also determined.4. The sense expression vector pCAM1301-CsRCA was introduced into cucumber inbredlines ‘08^-1’ with the agrobacterium-mediated method, and seven transgenic plants whichcontain two CsRCA copies were obtained （The wild type plants contain one CsRCA copy）.The transformation rate was about3.5%. The seven T0transgenic cucumber plants showed1¹.98fold in CsRCA mRNA abundance and a significant increase in expression of proteinlevel compared with the wild type （WT） plants.5. The diurnal variation of Pn and CsRCA mRNA expression of transgenic cucumberleaves in solar-greenhouse were single-peak pattern curve on Apr.17th （sunny day）. Theywere increased with the photon flux dension （PFD） and air temperature （Ta） increased, andthe peak appeared at12:00. The diurnal variations of CsRCA expression at the mRNA levelwere similar to that of Pn. This indicated that RCA participate in the diurnal variation ofphotosynthesis in cucumber leaves.6. CsRCA over-expression led to significant increase in the pigment contents，photosynthetic rate （Pn）, soluble sugar and starch contents in T1transgenic plant leavescompared with WT. T1transgenic plants also showed higher plant height, larger leaf area,more dry matter, earlier flowering time for1⁴d, and more ritio of female to male flowerthan WT.7. Low temperature stress led to decrease in CsRCA expression, Pn and stomatalconductance （Gs）, whereas increase in intercellular CO₂concentration （Ci） in cucumberleaves. The actual photochemical efficiency （ΦPSⅡ） and maximal photochemical efficiencyof PSⅡ in darkness （Fv/Fm） in cucumber seedlings also declined during chilling stress time,however the initial fluorescence （F0） increased. These data suggested that non-stomatallimitation was the main cause of the Pn decrease in cucumber seedlings during chilling stress.Chilling stress caused to injury in reactive center of PSⅡin the24h. Compared with the wildtype, the transgenic cucumber plants showed a lower decrease degree in CsRCA expression,Pn, Gs, ΦPSⅡ, and Fv/Fm, and a lower increase degree in Ci and F0. This data suggested thatover-expression of CsRCA alleviated the chilling damage degree in photosynthetic apparatusof cucumber seedlings, and maintained the relative stable photosynthesis.8. Low temperature stress resulted in a significant increase in malondialdehyde （MDA） content. The superoxidedismutase （SOD） activity increased rapidly at first6h, decreasedslowly at latter time during stress period. The peroxidase （POD） activity increased with thestress time, while the catalase （CAT） activity decreased. The ascorbate peroxidase （APX）activity decreased at the first6h, increased at the latter time during stress period. Theascorbic acid （AsA） and glutathione （GSH） contents showed the increasing trend. Incomparion with the wil-type, transgenic cucumber lines showed lower MDA content, higheractivities of SOD, POD, CAT and APX, and higher contents of AsA and GSH. The resultesrevealed that over-expression of CsRCA increased the ability of activated oxygen elimination,maintained the stability of membrane structure, and as the result, improved the chillingtolerance of cucumber seedlings.