Stress-resistance functional validation of transcription factor HaNAC12 of Haloxylon ammodendron
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摘要: 【目的】验证梭梭NAC转录因子基因（HaNAC12）的抗逆功能，以期解析梭梭响应逆境胁迫的分子机制，为梭梭及其他作物抗逆遗传改良提供理论参考。【方法】通过实时荧光定量PCR对HaNAC12基因在干旱、高盐、ABA处理下的表达模式分析。利用同源重组法、农杆菌介导喷花法、喷洒除草剂等方法构建并筛选HaNAC12转基因拟南芥阳性植株，在不同胁迫处理后测定其与野生型（WT）在萌发率、气孔开度、相对含水量、株高、生长速率和生理指标等方面的差异，并以正常生长的植株为对照，验证HaNAC12基因在拟南芥逆境胁迫下的抗逆功能。【结果】 HaNAC12基因相对表达量在干旱胁迫12 h时极显著高于0 h（P<0.01，下同），盐胁迫1 h时极显著高于0 h，ABA处理3和12 h均显著高于0 h （P<0.05，下同）。筛选获得3个高表达量的T3代转基因株系HaNAC12-1、HaNAC12-2和HaNAC12-3。种子干旱胁迫后WT的最终萌发率为45.00%，3个转基因株系的最终萌发率为66.11%~85.00%；种子盐胁迫后WT的最终萌发率为36.42%，3个转基因株系的最终萌发率为49.38%~58.64%。自然干旱和高盐胁迫下WT出现明显的干枯、枯黄现象，而3个转基因株系未出现干枯；自然干旱胁迫下3个转基因株系出现了提前抽薹、早花等生殖发育加速现象，且植株生长速率比WT快。苗期自然干旱胁迫后WT的气孔开度缩小67.54%，3个转基因株系的气孔开度分别缩小了91.08%、83.14%和85.04%，且HaNAC12-1、HaNAC12-2和HaNAC12-3株系的相对含水量显著或极显著高于WT。干旱和盐胁迫后3个转基因株系的丙二醛含量、脯氨酸含量、过氧化氢含量、过氧化氢酶活性均较胁迫前明显上升，其中，脯氨酸含量和过氧化氢酶活性上升幅度较WT大，而丙二醛含量和过氧化氢含量上升幅度较WT小。【结论】拟南芥过表达HaNAC12基因可增强拟南芥在萌发期和苗期对干旱和高盐胁迫的抗性，干旱胁迫可促进拟南芥开花，说明HaNAC12基因可提高转基因拟南芥的抗旱和耐盐性。Abstract: 【Objective】To verify the stress-resistance function of the NAC transcription factor gene（HaNAC12）of Haloxylon ammodendron，to analyze the molecular mechanism of H.ammodendron in response to stress，so as to provide theoretical reference for the genetic improvement of stress-resistance in H.ammodendron and other crops.【Method】Expression pattern of HaNAC12 gene under drought，salt and ABA treatments by quantitative real-time PCR（qRT-PCR）.HaNAC12 transgenic positive plants of Agrobacterium tumefaciens were cultivated and screened through homologous recombination method，A.tumefaciens-mediated flower spraying method and herbicide spraying method.Difference of germination rate，stomatal openness，relative water content，plant height，growth rate and physiological indexes after different stress treatments between the positive plants and wild type plant（WT）were determined and plants without treatments were taken as control to verify the stress-resistance function of HaNAC12 gene in A.tumefaciens under stresses.【Result】 Relative expression of HaNAC12 gene was extremely significantly higher at 12 h of drought stress than that at 0 h （P<0.01，the same below）.At 1 h of salt stress，relative expression of the gene was extremely significantly higher than that at 0 h.Relative expression of the gene was extremely significantly higher than that at both 3 and 12 h of ABA treatment（P<0.05，the same below）.Three high expression T3 generation transgenic lines HaNAC12-1，HaNAC12-2 and HaNAC12-3 were obtained by screening.Mean final germination rate of WT after seed drought stress was 45.00% and the final germination rate of the 3 transgenic lines was 66.11%-85.00%.Final germination rate of WT after seed salt stress was 36.42% and the final germination rate of the 3 transgenic strains was above 49.38%-58.64%.WT turned dry and yellow under natural drought and salt stress，while the 3 transgenic lines did not turn dry.The 3 transgenic lines showed seedling in accelerated development such as early bolting and early flowering under natural drought stress，and their growth rate was faster than WT.Stomatal opening was reduced by 67.54% in WT and 91.08%，83.14% and 85.04% in the three transgenic strains after natural drought stress at seedling stage，and relative water contents of HaNAC12-1，HaNAC12-2 and HaNAC12-3 strains were significantly or extremely significantly higher than that of WT.Malondialdehyde content，proline content，hydrogen peroxide content and catalase activity of the 3 transgenic strains increased significantly after drought and salt stress compared with those before stress，with greater increases of proline content and catalase activity than WT and smaller increases of malondialdehyde content and hydrogen peroxide content increasing than WT.【Conclusion】 Overexpression gene of the HaNAC12 in A.tumefaciens enhances its resistance to drought and salt stress at the germination and seedling stages，and promotes flowering in Arabidopsis after drought stress，indicating that HaNAC12 gene can improve the drought and salt tolerance of transgenic Arabidopsis.
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