The miR164-TaNAC14 module regulates root development and abiotic-stress tolerance in wheat seedlings

Previous studies have revealed the miR164 family and the miR164-targeted NAC transcription factor genes in rice(Oryza sativa)and Arabidopsis that play versatile roles in developmental processes and stress responses.In wheat(Triticum aestivum L.),we found nine genetic loci of tae-miR164(tae-MIR164 a to i)producing two mature sequences that downregulate the expression of three newly identified target genes of TaNACs(TaNAC1,TaNAC11,and TaNAC14)by the cleavage of the respective mRNAs.Overexpression of tae-miR164 or one of its target genes(TaNAC14)demonstrated that the miR164-TaNAC14 module greatly affects root growth and development and stress(drought and salinity)tolerance in wheat seedlings,and TaNAC14 promotes root growth and development in wheat seedlings and enhances drought tolerance,while tae-miR164 inhibits root development and reduces drought and salinity tolerance by downregulating the expression of TaNAC14.These findings identify the miR164-TaNAC14 module as well as other taemiR164-regulated genes which can serve as new genetic resources for stress-resistance wheat breeding.

小麦TaNAC基因基于可变剪切和microRNA的转录后调控分析

【目的】以条锈菌和白粉菌胁迫的普通小麦(Triticum aestivum L.)为研究对象,分析由可变剪切(alternative splicing,AS)形成的TaNAC结构变异转录本,同时分析TaNAC基因的microRNA调控位点,为进一步解析TaNAC基因通过转录后调控参与小麦响应真菌胁迫奠定基础。【方法】普通小麦兼抗种质N9134在被白粉菌和条锈菌分别侵染后,各8个时间点取样并混合,然后从混合样本中克隆得到大量TaNAC转录本。参考中国春小麦基因组注释信息(IWGSC RefSeq v1.1)进行比对,选择由可变剪切形成的TaNAC序列结构变异转录本,分析它们的序列结构特征。利用生物信息学软件和在线工具,对这些TaNAC结构变异转录本编码产物的功能结构域、高级结构、理化性质、亚细胞定位等特征和变异情况进行比对分析。同时,利用洋葱表皮细胞瞬时表达系统验证其中1对TaNAC结构变异转录本的亚细胞定位预测结果,并选取5组TaNAC基因的可变剪切序列结构变异转录本进行酵母转录自激活试验,研究序列结构变异对TaNAC基因转录调控活性的影响。此外,利用miRBase数据库收录的小麦中已报道的miRNAs和TaNAC基因,进行靶基因预测分析,建立小麦TaNAC家族成员与miRNAs的靶向关系。【结果】以条锈菌和白粉菌侵染的普通小麦兼抗种质N9134为材料,克隆得到的TaNAC转录本中的35条序列结构变异转录本由13个TaNAC基因可变剪切形成。通过分析发现,同一TaNAC基因由可变剪切形成的不同结构变异转录本的核酸序列结构存在差异,而且其对应编码产物的功能结构域、高级结构、理化性质和亚细胞定位等方面均会存在差异,同时也会表现出不同的转录调控活性;不同TaNAC基因的可变剪切方式存在差异,而且它们的结构变异转录本及其编码产物在结构特征、理化性质和转录调控活性等方面也均呈现出多样性的特征。通过分析TaNAC基因与其在编码序列区域的靶标tae-miRNAs,发现具有可变剪切转录本的TaNAC基因与tae-miRNA的结合位点均在其非可变剪切区域。【结论】TaNAC基因可以通过可变剪切这种转录后调控方式参与小麦对真菌胁迫的响应;同时发现,调控TaNAC基因的tae-miRNA可以独立于可变剪切这种转录后调控方式行使功能。

TaNAC14转基因小麦外源目标基因拷贝数及遗传稳定性分析

外源基因拷贝数是影响转基因植物遗传稳定性及其自身表达水平的重要因素。为了探析TaNAC14基因在小麦生长发育中的生物学功能,本研究通过农杆菌介导的遗传转化法获得了14个T0代TaNAC14转基因小麦植株,采用BASTA溶液涂抹和目标序列PCR检测相结合的方法,从14个转基因小麦植株中鉴定出9个阳性植株。通过TaqMan实时定量PCR方法,以小麦内源单拷贝基因Pinb为内参基因,对9个T0代转基因小麦阳性植株中外源目标基因拷贝数进行检测,结果表明,T0代转基因小麦再生植株中有5株为单拷贝,4株为双拷贝,其中单拷贝插入整合的比率接近55.6%。选取单拷贝转基因植株收获的种子进行种植,根据BASTA溶液涂抹鉴定对T0:1代小麦株系遗传情况进行分析,结果表明目标基因TaNAC14是可遗传的。此外,还对T1代转基因小麦目标基因表达水平进行测定,结果表明,与野生型JW1相比,各转基因小麦植株目的基因TaNAC14表达量均极显著增加。该研究结果为后续TaNAC14基因的功能研究奠定了基础。

普通小麦TaNAC1基因的表达及在拟南芥中的遗传转化

NAC类转录因子是植物特有的转录因子家族,在调节植物生长发育及逆境胁迫应答反应中起着重要作用。本文从普通小麦幼叶中获得了一个编码NAC结构域的转录因子基因,命名为Ta NAC1;氨基酸序列分析表明,Ta NAC1具有典型的NAC类转录因子所具有的五个亚结构域,隶属于NAC类转录因子的ATAF亚类;亚细胞定位实验表明,Ta NAC1蛋白在细胞核内表达;转录水平上,Ta NAC1基因的表达受到PEG、ABA、低温及高盐等非生物胁迫条件的诱导;将Ta NAC1转化拟南芥后,与野生型比较发现,Ta NAC1基因的过量表达会使转基因植株出现叶片发育畸形且生长缓慢,植株矮化及茎部融合等表型,表明Ta NAC1基因可能在参与小麦叶片及茎的发育中起着重要的调控作用。

TaNAC48 positively regulates drought tolerance and ABA responses in wheat(Triticum aestivum L.)

NAC family transcription factors(TFs) are important regulators in plant development and stress responses. However, the biological functions of NAC TFs in wheat are rarely studied. In this study, 43 putative drought-induced NAC genes were identified from de novo transcriptome sequencing data of wheat following drought treatment. Twelve wheat NACs along with ten known stress-related NACs from Arabidopsis and rice were clustered into Group II based on a phylogenetic analysis. Ta NAC48, which showed a higher and constitutive expression level in Group Ⅱ, was selected for further investigation.Ta NAC48 transcript was up-regulated by drought, PEG, H_(2)O_(2) and abscisic acid(ABA) treatment and encoded a nuclear localized protein. Overexpression of Ta NAC48 significantly promoted drought tolerance with increased proline content, and decreased rates of water loss, malondialdehyde(MDA), H_(2)O_(2) and O_(2)^(-) content. Root length and a stomatal aperture assay confirmed that Ta NAC48-overexpression plants increased sensitivity to ABA. Electrophoretic mobility shift assay(EMSA) and luciferase reporter analysis indicated that Ta AREB3 could bind to a cis-acting ABA-responsive element(ABRE) on Ta NAC48 promoter and activate the expression of Ta NAC48. These results suggest that Ta NAC48 is essential in mediating crosstalk between the ABA signaling pathway and drought stress responses in wheat.

小麦NAC转录因子TaNAC14的原核表达和分离纯化

植物NAC转录因子参与调控多种生物学过程,在植物生长发育和逆境适应性方面发挥重要作用。前期研究发现小麦(Triticum aestivum)NAC家族成员TaNAC14正向调控幼苗根生长发育并增强其耐旱性。本研究对小麦TaNAC14蛋白的理化性质和结构进行了分析预测;对TaNAC14的亚细胞定位和转录激活活性进行了验证;构建了TaNAC14重组蛋白原核表达载体pET21a-HMT-TaNAC14,转化大肠杆菌(Escherichia coli)表达菌株BL21 CodonPlus(DE3)-RIPL,对TaNAC14重组蛋白的诱导表达条件进行了优化,对重组蛋白的可溶性进行了分析,并利用镍柱对重组蛋白进行了分离纯化。结果表明,TaNAC14具有NAM家族保守的结构域,定位于细胞核,具有转录激活活性。重组蛋白HMT-TaNAC14在大肠杆菌中的最佳诱导表达条件为0.2mmol/L的IPTG诱导4 h;该重组蛋白主要以可溶性形式存在;利用镍柱对其进行了分离纯化,获得了较高纯度的目的蛋白。本研究为后续进行TaNAC14调控靶基因的鉴定奠定了基础。

Variation in cis-regulation of a NAC transcription factor contributes to drought tolerance in wheat

Drought is a major environmental factor limiting wheat production worldwide,and developing drought-tolerant cultivars is a central challenge for wheat breeders globally.Therefore,it is important to identify genetic components determining drought tolerance in wheat.In this study,we identified a wheat NAC gene(TaNAC071-A)that is tightly associated with drought tolerance by a genome-wide association study.Knockdown of TaNAC071-A in wheat attenuated plant drought tolerance,whereas its overexpression significantly enhanced drought tolerance through improved water-use efficiency and increased expression of stress-responsive genes.This heightened water-saving mechanism mitigated the yield loss caused by water deficit.Further candidate gene association analysis showed that a 108-bp insertion in the promoter of TaNAC071-A alters its expression level and contributes to variation in drought tolerance among wheat accessions.This insertion contains two MYB cis-regulatory elements(CREs)that can be directly bound by the MYB transcription activator,TaMYBL1,thereby leading to increased TaNAC071-A expression and plant drought tolerance.Importantly,introgression of this 108-bp insertion allele,TaNAC071-AIn-693,into drought-sensitive cultivars could improve their drought tolerance,demonstrating that it is a valuable genetic resource for wheat breeding.Taken together,our findings highlight a major breakthrough in determining the genetic basis underlying phenotypic variation in wheat drought tolerance and showcase the potential of exploiting CRE-containing indels for improving important agronomical traits.