摘要
玉米是世界上重要的粮食作物之一，干旱、盐渍、低温等非生物胁迫对玉米的生长造成了严重的影响，也是限制其产量的主要胁迫因素，抗逆育种一直是重要的育种目标。挖掘抗逆相关功能基因并利用这些基因培育抗逆新品种是应对逆境胁迫，提高玉米产量的最为有效途径。研究表明，转录因子在提高作物对胁迫的耐受性过程中起到了重要作用。过量表达特定的胁迫应答转录因子可以调控多个下游抗逆功能基因的同时表达，从而使作物获得良好的综合改良效果。目前，这种方法已经成为植物抗逆遗传改良育种中的研究热点。同源异型-亮氨酸拉链(HD-Zip)蛋白是高等植物特异的一类转录因子，参与了植物的胁迫应答和生长发育调控。因此，挖掘胁迫应答HD-Zip基因对开展玉米抗逆改良育种具有重要的意义。本研究运用生物信息学的方法对玉米全基因组HD-Zip基因进行鉴定，分析这些基因的结构特点、进化关系以及干旱诱导表达模式，并通过过量表达技术对玉米Zmhdz10基因在植物非生物胁迫应答过程中的功能及作用机制进行了研究，获得了如下主要结果：
1.通过Blast同源搜索，共鉴定了55个非重复的玉米HD-Zip基因家族成员并从基因结构和进化等方面进行了分析。研究发现，玉米HD-Zip家族的大部分基因序列高度保守，与拟南芥和水稻HD-Zip基因在结构上不存在明显差异。根据进化关系，55个玉米HD-Zip基因被进一步分为4个亚族(I-IV)。染色体定位分析显示，55个基因在10条染色体上的分布是非随机的。对玉米HD-Zip基因复制事件进行分析，发现参与片段复制的基因占玉米HD-Zip基因总数的62%，表明片段复制对玉米HD-Zip基因数量的扩张起到了重要作用。
2.对17个HD-Zip I基因上游2kb启动子序列的抗逆相关顺式作用元件(ABRE，LTRE和DRE)进行了分析，发现除Zmhdz16外，其余16个基因的启动子区域均含有1到多个抗逆相关顺式作用元件。对复制基因启动子区域内的3种顺式作用元件的分布进行比较，发现三种顺式作用元件在复制基因启动子区域内的数量和位置有较大的差异。利用荧光定量PCR方法对HD-Zip I基因的干旱诱导表达模式进行了分析，发现有12个基因的表达显著上调，其它5个基因表达下调。对复制基因的干旱诱导表达模式进行比较，发现复制基因的表达模式总体较为相似，说明这些基因可能存在着基因功能的冗余现象。
3.根据进化关系和干旱诱导表达模式，筛选了一个受干旱强烈诱导表达的候选基因Zmhdz10并从玉米自交系B73中克隆了该基因。序列分析显示，Zmhdz10基因cDNA全长为825bp，编码274个氨基酸，与玉米B73数据库中公布的序列完全一致。进一步的表达分析发现Zmhdz10的表达还受到盐和外源ABA的诱导。组织表达模式分析显示，Zmhdz10在根、茎、叶、果穗、雄花序、幼芽和花丝7个组织中均有表达，其中在叶中的表达量最高。通过基因枪轰击洋葱表皮细胞的瞬时表达分析显示，Zmhdz10-GFP融合表达蛋白定位在细胞核中。酵母杂交实验表明，Zmhdz10在酵母细胞中能够激活报告基因的表达，能够对反向重复序列CAAT(A/T)ATTG发生特异性的结合。
4.过量表达Zmhdz10的转基因植株在生长表型上与对照植株没有明显差异。在干旱和盐胁迫处理后，Zmhdz10转基因水稻表现出对胁迫较强的耐受性。通过对相对电解质渗透率、丙二醛含量和脯氨酸含量等生理生化指标进行测定，发现转基因植株在胁迫条件下的相对电解质渗透率和丙二醛含量低于野生型植株，脯氨酸含量高于野生型植株。另外，ABA敏感性实验显示Zmhdz10过量表达转基因植株显著提高了对外源ABA的敏感性。
5. Zmhdz10过量表达的转基因拟南芥植株同样表现出对干旱和盐胁迫较强的适应性和忍耐力。通过对P5CS1、RD22、RD29B和ABI14个胁迫/ABA应答功能基因的表达分析发现，在干旱条件下，这些抗逆功能基因在转基因拟南芥中的表达量显著高于野生型植株，表达被激活。对参与非依赖ABA信号传导途径的ERD1表达分析显示，该基因在野生型和转基因植株中的表达没有显著差异。
综上所述，本研究通过Blast同源搜索，在玉米全基因组水平上共鉴定了55个HD-Zip基因。根据基因结构、进化和干旱诱导表达模式等分析结果，克隆了Zmhdz10基因并对其分子生物学特性进行分析。过量表达该基因显著提高了转基因植株耐旱和耐盐性，为利用基因工程的方法改良作物的抗逆性提供了优良的基因资源。同时，转基因植株对ABA的敏感性的增加和胁迫/ABA应答功能基因的表达上调，表明该基因主要通过ABA信号传导途径调控抗逆功能基因的表达，从而提高转基因植株的耐旱和耐盐性。
Maize is one of the most important crops in the world. Environmental stresses,such as drought, salinity and low temperature, have adverse effects on maize growthand yield. Adversity-resistance breeding has been the important breeding objectives.To gain stress tolerance-related functional genes and cultivate new stress-tolerantcrops using these genes is one of the most effective measures to respond abiotic stressand increase maize yield. Stuides showed that transcription factors have importantfunctions on improving crop tolerance to various stresses. Overexpression of aspecific transcription factor related to stress response can regulate the expression ofmultiple downstream stress-responsive genes at the same time and consequentlyimprove comprehensive resistances of crops. At present, this method has become aresearch hotspot in the genetic improvement of plant resistance breeding.Homeodomain-leucine zipper (HD-Zip) proteins are unique to plants and have diversefunctions in regulating plant growth and stress response. Thus, isolating HD-Zipgenes related to stress response has important significance for maizeadversity-resistance breeding. In this study, we performed genome-wide survey ofHD-Zip genes in maize, and characterized their structure features, phylogeneticrelationships and drought-induced expression patterns. We subsequently study thefunctions of Zmhdz10gene in abiotic stress response by overexpressing this gene inplants. The main results are as follows:
1. By Blast homologous search, a total of55non-redundant HD-Zip genes wereidentified in the maize genome and characterized for their structure and evolution, etc.Results showed that most of the genes have highly conserved sequences, and therewas no significant difference in their structure among different plant species. The55HD-Zip genes were further divided into four classes (I-IV) based on phylogeny.Chromosomal location of these genes revealed that they are distributed unevenlyacross all the10chromosomes. Analysis of gene duplication events showed thatsegmental duplicated genes accounted for62%of the maize HD-Zip genes,suggesting that segmental duplication play an important role in the expansion ofmaize HD-ZIP gene family.
2. Three types of stress-related cis-elements, including ABRE, LTRE and DREwere identified in the2kb promoter regions upstream of the transcription start site ofthe17HD-Zip I genes. Results showed that all the HD-Zip I genes except for Zmhdz16contained one or more stress-related cis-elements in their promoter regions.By comparing the distribution of the three regulatory elements in the promoter regions,the duplicated genes were found to exhibit significant differences in their promotersequences. Expression levels of the HD-Zip I genes under drought treatment wereinvestigated by quantitative real-time PCR. Of the17HD-Zip I genes, the expressionsof12genes were obviously up-regulated, whereas those of the other5genes weredown-regulated. Expression profiles of the duplicated genes were also compared, andthe results revealed that the duplicated genes exhibited the similar expression patternsunder drought treatment.
3. According to phylogenetic relationships and drought-induced expressionpatterns, a drought strongly indued gene, Zmhdz10, was cloned from maize inbredline B73. Sequence analysis showed that the full-length cDNA of Zmhdz10is825-bp,which encodes a protein of274amino acids. The cDNA sequence of Zmhdz10shares100%sequence identity to the predicted sequence in the maize B73annotateddatabase. Further expression analysis showed that Zmhdz10was also induced by saltstress and ABA, and tissue-specific expression indicated that Zmhdz10wasconstitutively expressed in roots, stems, leaves, tassels, ears, silks and seedlings, butthe highest expression was detected in leaves. Transient expression showed that theGFP signal was detected only in the nucleus of the onion cell for Zmhdz10-GFPfusion protein using bombarding transformation. Yeast hybrid assays showed thatZmhdz10can activate the expression of the reporter genes, and specifically bind tothe pseudopalindromic sequence CAAT(A/T)ATTG.
4. Zmhdz10overexpression transgenic plants have no significant differences inthe morphology compared with control plants. After drought and salt treatments,Zmhdz10overexpression transgenic plants showed significantly enhanced tolerance todrought and salt stresses. Relative electrolyte leakage, malondialdehyde and prolinewere tested in WT and transgenic plants. Results showed that relative electrolyteleakage and malondialdehyde content in Zmhdz10overexpression plants weresignificantly lower than that in WT plants under stressed conditions, but prolinecontent was higher than that in WT plants. In addition, ABA sensitivity assayindicated that Zmhdz10overexpression transgenic plants exhibit significantlyincreased sensitivity to ABA.
5. Zmhdz10overexpression Arabidopsis plants also showed significantlyimproved tolerance to drought and salt stresses. After drought treatment, expression levels of the tested stress/ABA-responsive marker genes, including P5CS1, RD22,RD29B and ABI1, were significantly higher than detected in WT plants, suggestingthat expression of these genes were actived in transgenic plants. No significantdifference in the expression of ERD1was found between WT and transgenic plantsafter drought treatment, which was proved to involve in the ABA-independentpathways as described previously.
In conclusion, a total of55HD-Zip genes were identified in the maize genomeby Blast homologous search. According to the analysis of gene structure, phylogeneticrelationships and drought-induced expression patterns, Zmhdz10gene was cloned, andits molecular biological characteristics were also analyzed in this study.Overexpression of Zmhdz10could significantly increase tolerance to drought and saltstresses, which provides an excellent gene resource for improving crop resistancethrough genetic engineering methods. Moreover, the increased sensitivity to ABA andup-regulated expression of stress/ABA-responsive functional genes of transgenicplants indicate that Zmhdz10can regulate the expression of stress tolerance-relatedfunctional genes in response to abiotic stress through the ABA signal transductionpathway.
引文
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