黄瓜CsPLDα基因对NaC1胁迫的响应研究

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土壤次生盐渍化是温室蔬菜生长的主要障碍之一,严重制约设施蔬菜的可持续高效发展。黄瓜作为我国设施主栽蔬菜之一,属于盐敏感植物,土壤次生盐渍化危害尤为严重。磷脂酶D(PLD)是植物中的一种主要磷脂水解酶,其水解产物如IP3、DAG、PA和乙醇胺等还可作为第二信使,参与信号传递过程,触发细胞内与抗性有关的一系列反应,参与生长发育和各种生物、非生物胁迫信号转导。因此,通过基因工程手段获得过表达CsPLDa植株,并研究其在NaCl胁迫中的功能,为创制黄瓜耐盐新种质和克服土壤次生盐渍化提供理论依据。本课题在已有研究基础上,通过农杆菌介导法分别获得过表达CsPLDa烟草和黄瓜植株;并通过水培方法,研究了NaCl胁迫对黄瓜幼苗CsPLDa表达及生理生化特性的影响,以期探究黄瓜CsPLDa对NaCl胁迫的响应机制。取得主要结果如下:1.利用农杆菌侵染法将pBI121-CsPLDa(+)转入黄瓜,对T0代转基因黄瓜进行PCR检测,共获得5株正义转基因植株。将转基因黄瓜T0代播种自交留种,对T1代5个株系的幼苗进行PCR检测,幼苗检测呈阳性;从中任意挑选出3个株系,进一步Real-time PCR检测结果发现,CsPLDa已整合到黄瓜基因组中且已表达。2.利用农杆菌侵染法将pBI121-CsPLDa (+)转入烟草,得到98株烟草幼苗,经T0代转基因烟草通过PCR检测表明,共获得90株转基因株系;从中任意挑选5株,经Real-time PCR检测表明,CsPLDa已插入烟草基因组中且已表达。其中挑选出2个表达量高的株系进行种子抗盐性试验,结果表明,200mM NaCl处理下培养9d后,转基因烟草种子T1-68和T1-71发芽率分别高达95%和92.5%,而野生型仅为30%左右,说明转基因烟草种子明显提高了对NaCl胁迫的抗性。3. NaCl处理后,黄瓜幼苗叶片中CsPLDa、CsNMAPK表达和NO含量均有激发现象;且CsPLDa表达量和NO含量均在处理2h后达最大值;而CsNMAPK表达在处理0.5h后达到最高,其后均呈降低趋势。以上结果表明,黄瓜幼苗叶片中CsPLDa基因表达滞后于CsNMAPK基因表达,且能激发NO的产生。4. NaCl处理后,黄瓜幼苗叶片中H2O2和O2-含量均随处理时间延长呈明显上升趋势。而叶片中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)和抗坏血酸过氧化物酶(APX)活性变化趋势一致,与处理Oh相比,均在0.5~2h内呈先降低后升高趋势,但处理4~24h后均呈逐渐升高趋势。5.200mM NaCl处理0.5~2h后,叶片中丙二醛(MDA)、脱落酸(ABA)及可溶性蛋白(Pro)含量均显著低于0h;而后呈升高趋势,均在处理24h后达最大值。叶片中赤霉素(GA)和水杨酸(SA)含量均呈先下降后上升又下降趋势;处理4h后,SA含量达最大值;处理24h后其叶片中SA和GA含量均显著低于0h。
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