PagHK3a基因敲除对银腺杨抗旱性的影响

Effects of PagHK3a Gene Knockout on Drought Resistance of Populus alba×P.glandulosa

[目的]:对银腺杨(Populus alba×P.glandulosa‘84k’)组氨酸激酶PagHK3a基因敲除株系的抗旱性进行评价,同时探究PagHK3a基因在杨树响应干旱胁迫过程中的分子调节机制。[方法]:利用5%PEG模拟干旱胁迫处理继代25 d的野生型银腺杨(WT)及其PagHK3a基因敲除株系(C1和C2)组培苗,处理3 h后采用实时定量PCR(Quantitative real-time PCR)方法测定各株系叶片PagHK3a基因、PagHK3a下游基因、干旱胁迫响应基因及抗氧化基因的表达水平;利用称重控水法对各株系盆栽苗进行3个梯度的温室干旱胁迫处理,包括正常浇水(土壤相对含水量:70%~75%)、中度干旱(40%~45%)以及重度干旱(25%~30%),干旱胁迫4周后测定WT及C1、C2株系的瞬时光合参数、过氧化氢(H_(2)O_(2))及丙二醛(MDA)含量、过氧化物酶(POD)及超氧化物歧化酶(SOD)酶活性、株高和地径等生理生化及生长指标。[结果]:在温室中度干旱条件下,C1和C2株系株高生长量均显著高于WT,而在重度干旱条件下,这两个株系的株高生长量与WT相比均无显著差异。分析光合参数发现,在中度干旱条件下,基因敲除株系C1和C2气孔导度及胞间CO_(2)浓度均显著高于WT,但在重度干旱胁迫条件下只有C2株系胞间CO_(2)浓度显著高于WT;在中度干旱胁迫条件下C1株系瞬时净光合速率显著高于WT,在重度干旱胁迫条件下,C2株系瞬时净光合速率显著高于WT。同时生化指标测定结果显示,在中度干旱条件下基因敲除株系C1和C2中MDA及H_(2)O_(2)含量显著低于WT,而在重度干旱胁迫时,仅C2株系显著低于WT;对比分析各株系抗氧化酶活性发现,正常供水条件下,C1株系叶片POD酶活性以及C2株系SOD酶活性显著高于WT,在中度干旱胁迫条件下,C1株系这两种保护酶活性均显著高于WT,而C2株系仅在重度干旱胁迫下显著高于WT。另外,在PEG模拟干旱胁迫处理下,基因敲除株系C1和C2叶片组氨酸激酶基因PagHK3a及其下游响应调节蛋白PagRR2、PagRR15基因表达量与WT相比均显著下调;而干旱胁迫响应基因PagNAC3以及过氧化物酶合成基因POD1的表达则显著上调,超氧化物歧化酶合成基因SOD4的表达与WT相比无显著差异。[结论]:在PagHK3a基因敲除株系中,PagHK3a基因及细胞分裂素信号传导途径中响应调节因子基因PagRR2、PagRR15表达均较WT显著降低,而胁迫响应基因PagNAC3及POD合成基因POD1表达显著升高;同时,在中度干旱条件下PagHK3a基因敲除株系的气体交换能力更强,MDA以及H_(2)O_(2)含量更低,株高生长量更大,从而具有更强的抗旱能力。

[Objective]:This study aimed to evaluate the drought resistance of two histidine kinase PagHK3a gene knockout lines of poplar'84k'(Populus alba×P.glandulosa'84k'),and explore the molecular regulatory mechanism of PagHK3a gene in poplar response to drought stress.[Method]:Plantlets of wild-type(WT)and gene knockout strains(C1 and C2)sub-cultured for 25 days were treated with 5%PEG and the mature leaves of the plantlets were gathered 3 hours later.Quantitative real-time PCR(qPCR)was used to detect the expression levels of PagHK3a gene,PagHK3a downstream related genes,drought stress response genes and antioxidant related genes in leaves of the three lines.Meanwhile,the drought stress treatment was carried out using the potted plants of each line by weighing method with three levels:normal watering greenhouse(soil relative water content:70%-75%),moderate drought(40%-45%)and severe drought(25%-30%).After four weeks of drought stress treatment,transient photosynthetic parameters,contents of hydrogen peroxide(H_(2)O_(2))and malondialdehyde(MDA),activities of peroxidase(POD)and superoxide dismutase(SOD),plant height and ground diameter of WT and C1,C2 were measured.[Result]:Under moderate drought conditions,the height growth of both C1 and C2 lines was significantly higher than that of WT,but under severe drought conditions,the height growth of both lines was not significantly different from that of WT.Analysis of photosynthetic parameters revealed that the stomatal conductance and intercellular carbon dioxide concentration of C1 and C2 were significantly higher than those of WT under moderate drought condition,but only C2 was significantly higher than that of WT under severe drought stress condition.Under moderate drought stress,the instantaneous net photosynthetic rate of C1 was significantly higher than that of WT,and under severe drought stress,the instantaneous net photosynthetic rate of C2 was significantly higher than that of WT.At the same time,the results of biochemical indexes showed that MDA and H_(2)O_(2)contents in knockout lines C1 and C2 were significantly lower than those in WT under moderate drought stress,but only C2 was significantly lower than that in WT under severe drought stress.Comparative analysis of the antioxidant enzyme activity of each line found that,under normal water supply conditions,the POD enzyme activity of C1 strain and the SOD activity of C2 strain were significantly higher than that of WT.And under moderate drought stress,the two protective enzyme activities of C1 strain were significantly higher than that of the control,while C2 was significantly higher than that of WT only under severe drought stress.In addition,under 5%PEG treatment,the expression levels of histidine kinase gene PagHK3a and its downstream response regulatory proteins PagRR2 and PagRR15 in the leaves of gene knockout lines C1 and C2 were significantly down-regulated compared with those of WT.The expression levels of drought stress response gene PagNAC3 and peroxidase synthesis gene POD1 were significantly up-regulated,while the expression level of superoxide dismutase synthesis gene SOD4 was not significantly different from that of WT.[Conclusion]:In PagHK3a knockout lines,expression of PagHK3a was down-regulated,expression of its downstream genes PagRR2 and PagRR15 in the cytokinin signaling pathway was also significant down regulated and the expression of stress response gene PagNAC3 and POD synthesis gene POD1 were significantly up regulated;meanwhile the PagHK3a knockout lines had stronger gas exchange ability,lower MDA and H_(2)O_(2)content and larger plant height growth under moderate drought conditions,therefore had stronger drought stress resistance than WT.