玉米ZmCCT10耐低氮功能研究

Investigation of Low Nitrogen Tolerance of ZmCCT10 in Maize

【目的】土壤氮素缺乏影响玉米的产量和品质,是中国玉米生产面临的重大问题。ZmCCT10编码转录因子,具有一因多效性,ZmCCT10是调控玉米生长发育和响应非生物胁迫重要的调节因子。解析玉米耐低氮的分子机制、聚合抗性基因,为培育耐低氮和氮高效玉米品种奠定基础。【方法】通过比较ZmCCT10近等基因系在低氮胁迫和完全营养水培条件下与耐低氮胁迫相关的性状,分析低氮胁迫后ZmCCT10的表达模式,选择ZmCCT10在近等基因系表达量差异最大的部位与时间点,进行转录组测序。发掘玉米ZmCCT10响应耐低氮反应的特征,探究其参与耐低氮反应的分子机制。【结果】在低氮胁迫条件下,ZmCCT10近等基因系Y331-ΔTE和Y331的根长性状、生物量、氮素生理指标显著差异。其中,ZmCCT10不带有转座子插入的单倍型Y331-ΔTE的总根长、主胚根长、侧根长均显著长于Y331;根干重、地上部干重、氮积累量、硝酸还原酶活性也显著高于Y331。在低氮胁迫后,ZmCCT10在根部和叶片的表达量均显著高于对照处理,且近等基因系间的表达量也具有显著差异,根部和叶片的表达模式也不同。胁迫处理3 h后,ZmCCT10在根部的表达量达到峰值,而在叶片的表达量随胁迫处理时间的增加而持续升高,在胁迫处理后6 h达到峰值。选取0.04 mmol·L^(-1)低氮胁迫处理3 h的根部样品进行转录组测序,生物学重复间的相关系数达0.9以上。GO富集分析表明,低氮胁迫后,参与胺化物合成过程和细胞氮化物代谢过程的基因在近等基因系间的表达量差异显著。结合差异基因的表达量和表达模式,筛选ZmCCT10调控参与玉米耐低氮反应的候选基因。经qRT-PCR验证,转录组测序获得的与耐低氮相关的ZmMPK5、ZmNS2等基因在近等基因系胁迫前后的表达量差异显著。【结论】ZmCCT10是一个以转录调控的方式参与玉米耐低氮反应的候选基因。

【Objective】The lack of soil nitrogen impacts the yield and quality of maize,which is a major problem of maize production in China.ZmCCT10 encodes the transcription factor,which is pleiotropic.ZmCCT10 is a very important co-factor regulating the growth,development and responding to abiotic stress of maize.The molecular mechanism of maize tolerance to low nitrogen is the basis for breeding maize varieties with low nitrogen tolerance and high nitrogen efficiency.【Method】In this study,we compared the traits those relate to low-nitrogen tolerance,expression pattern of ZmCCT10 and transcriptome results of ZmCCT10 near-isogenic lines under low-nitrogen stress and complete nutrient conditions.To analysis the characteristics of ZmCCT10 in response to low-nitrogen stress and the molecular mechanisms involved in low-nitrogen tolerance were explored.【Result】This study indicated that different alleles of ZmCCT10 showed significant differences in root length traits,biomass and nitrogen physiological traits under low nitrogen stress.The Y331-ΔTE haplotype without transposon insertion of ZmCCT10 had significantly longer total root length,main radicle length and lateral root length than Y331 after low nitrogen stress.What is more,root dry weight,shoot dry weight,nitrogen accumulation and nitrate reductase activity were also significantly higher than Y331.The expression levels of ZmCCT10 in roots and leaves were significantly higher than those in the control treatment.The expression level of ZmCCT10 in roots reached a peak at 3 hours after stress treatment.In leaves,the expression of ZmCCT10 continued to increase and peaked 6 hours after stress treatment.Root samples were collected under 0.04 mmol·L^(-1) low nitrogen stress after 3h for transcriptome sequencing.The correlation coefficients between biological replicates are more than 0.9.GO enrichment analysis showed that the expression levels of amine synthesis process and cellular nitrogen compound catabolic process were significantly different in near-isogenic lines after low nitrogen stress.Combined with the amount and expression pattern of differential genes,ZmCCT10-regulated candidate genes involved in low-nitrogen tolerance were selected.qRT-PCR confirmed that the expression levels of ZmMPK5,ZmNS2 and other genes were significantly different after stress in near-isogenic lines.【Conclusion】ZmCCT10 is a candidate gene involved in low nitrogen tolerance in maize and it participates in the low-nitrogen tolerance response of maize as transcriptional regulation.