干旱、盐碱、低温和冻害等非生物逆境是影响植物生长发育的主要环境因素，严重地制约着农作物产量与品质的提高。在逆境胁迫下，植物能感知、传递胁迫信号，通过激活响应转录因子，启动抗逆功能基因的表达，使植物体内发生一系列的生理生化反应，抵御不良环境的危害，从而提高植物的耐逆性。 NAC转录因子基因家族是植物所特有的基因家族，也是植物中最大的基因家族之一。NAC转录因子最初来源于矮牵牛NAM基因和拟南芥ATAF1，ATAF2和CUC基因的一段共有的序列。近年来，通过对大量NAC型转录因子基因的功能和特征分析证明该家族基因在植物的胁迫应答过程中起着重要的作用。碱性/螺旋-环-螺旋(bHLH)转录因子家族是一个在动植物中广泛存在的功能多样性的基因家族。近年来，从已经获得全基因组序列的物种中鉴定了几百个bHLH型转录因子基因。一些植物的bHLH型转录因子也被鉴定和特征分析，其功能涉及生长发育以及胁迫响应等。然而，bHLH型转录因子家族的大多数成员仍有待于研究。 棉花是世界性的重要经济作物，其中陆地棉占全球棉花种植面积的95%以上，然而在其生长区域频繁遭受着干旱、高盐、低温等非生物胁迫的侵袭，严重影响着棉花的生长发育和产量的提高。本研究选用我国生产上广泛利用的抗逆品种陆地棉晋棉19为试验材料，选择了与胁迫相关的NAC型以及bHLH型转录因子基因进行研究，以期获得在作物抗逆性改良方面具有应用潜力的候选转录因子基因，对棉花的抗逆育种以及阐述棉花耐受胁迫的分子机制都有着重要的意义。 本研究取得了以下研究进展： 首次从陆地棉晋棉19中分离到了6个NAC型胁迫相关转录因子GhNAC1- GhNAC6。在拟南芥中，NAC基因在非生物胁迫的信号调控中起着重要的作用。我们把这个结果拓展到了棉花上，利用拟南芥的NAC基因氨基酸序列为搜索探针扫描陆地棉EST数据库，重叠候选的ESTs，我们从陆地棉中获得了6个推测的编码NAC型蛋白的全长cDNA序列。根据推测的这6个全长cDNA序列，我们设计了巢式PCR引物对和横跨全长ORF的PCR引物对，然后将这6个NAC型胁迫相关的转录因子GhNAC1-GhNAC6从陆地棉晋棉19的叶片中克隆。这六个全长的cDNA序列(GhNAC1-GhNAC6)，每个都含有一个单个完整的ORF，分别编码276, 299, 298, 346, 356 和 327个氨基酸，推测的分子量分别为31.9, 33.8, 33.9, 38.4, 40.2和37.0 KDa，等电点分别为5.83, 5.74, 6.54, 8.87, 8.17 和5.39。这六个基因都具有保守的内含子/外显子结构，然而具有不同的内含子长度和染色体位置。GhNAC1-GhNAC6序列具有很高的相似性，特别是在NAC结构域区。半定量RT-PCR分析表明，这六个基因都能够在叶片中丰富的表达，而在根、茎和纤维中微量的表达或者不表达。实时定量RT-PCR分析表明，这六个基因不同程度的受到了干旱、高盐、低温和ABA的诱导。根据胁迫诱导的NAC蛋白系统进化树分析结果，我们鉴定的这6个陆地棉NAC蛋白分布在这5个亚家族中的4个，分别为ATAF，AtNAC3，NAP 和NAM，它们中的3个亚家族，ATAF，AtNAC3和NAP类NAC基因蛋白已在大量植物中被证明参与生物与非生物环境的胁迫响应。 首次从陆地棉晋棉19中分离到了bHLH型胁迫相关的转录因子GhbHLH1。众所周知，植物激素ABA在调控植物响应干旱胁迫中起着重要的作用，AtMYC2 (和GhbHLH1同源)已经被证明其作为转录因子涉及拟南芥的干旱和ABA应答通路。本研究用AtMYC2的氨基酸序列为搜索探针扫描陆地棉EST数据库，重叠候选的ESTs，拼接出一个新的棉花bHLH转录因子的cDNA序列。根据这个拼接的cDNA序列，设计了一对横跨这个转录因子全长ORF的特异引物。最终，我们从胁迫诱导的陆地棉晋棉19的叶片cDNA中，利用RT-PCR的方法分离到了这个可能在棉花中调控ABA应答的重要基因(GhbHLH1)。全长GhbHLH1的cDNA序列有2025bp的开放阅读框，编码674个氨基酸，推测的分子量和等电点分别为73.6 kDa和5.46。序列比对表明，GhbHLH1蛋白含有一个60个氨基酸长的bHLH结构域，其氨基酸序列和拟南芥的bHLH转录因子有很高的同源关系，特别是和在胁迫反应中起重要作用的AtMYC2。半定量RT-PCR分析表明，GhbHLH1基因能够在根、茎、叶和开花7天后的纤维中组成性的表达，特别是在开花7天后的纤维中表达量最强。虽然GhbHLH1基因能够组成性的在根、茎、叶和开花7天后的纤维中表达，但是实时荧光定量RT-PCR表明在叶片中GhbHLH1的表达能够短暂的被ABA和PEG的处理所诱导，而GhbHLH1的表达却不被高盐和低温影响。GhbHLH1基因的进一步功能验证正在进行中。

Drought, salinity, cold and freeze are the key environment factors affecting plant growth and development, and seriously limit the increase of yield and improvement of quality of crops. Under abiotic stress, plants could sense and transduce the signals caused by stress to transcription factors through a series of phosphorization responses. The functional genes related to stress tolerance are then initiated; a series of physio- chemical reactions are activated to alleviate the damage caused by abiotic stress, finally the stress tolerance of plants is enhanced. NAC transcription factor family genes are plant specific transcriptional regulators, and this gene family is one of the biggest gene families in plant. The NAC domain was originally characterized from consensus sequences from petunia NAM and from Arabidopsis ATAF1, ATAF2, and CUC2. NAC transcription factor genes play very important roles in stress response. The basic helix-loop-helix (bHLH) proteins are a group of functionally diverse transcription factors found in both plants and animals. Recently, hundreds of bHLH genes were identified in organisms whose genome sequences were available. Several plant bHLH-type proteins have been characterized, which act as development and growth and stress response. However, the biological roles of most members of this gene family in plant remain to be studied. Cotton (Gossypium spp.) is a major cash crop for both textiles and food. Upland cotton (Gossypium hirsutum L.) accounts for >95% of world cotton production. However, cotton-growing areas are subject to extreme drought, salinity, and temperature, each of which can impede cotton growth and production. Our research focused on identifying the NAC type and bHLH type ranscription factor genes involved in abiotic from Gossypium hirsutum L cv Jinmian 19(a widely used stresses- resistant cultivar in china for breeding) and providing new targets for producing tolerance-enhanced transgenics. It is of important significance to cotton stress-tolerant breeding and clarification of stress-tolerant molecular mechanism. Progresses of this research are following: We identified six abiotic stress related NAC-type transcription factors (GhNAC1- GhNAC6) for the first time in cotton. In A. thaliana, NAC-domain proteins contribute to abiotic stress signal transduction pathways. We extend these results to cotton. NAC domain protein sequences from A. thaliana were used as queries to screen the G. hirsutum L. EST database, and contigging the candidate ESTs. We obtain six putative full-length cDNA sequences encoding NAC-like proteins in cotton. We designed Nested-PCR prime pairs and PCR prime pairs of full-length ORF based on this six putative cDNA sequences, then six abiotic stress related NAC-type transcription factors (GhNAC1-GhNAC6) were isolated from leaves cDNA of upland cotton cv Jinmian 19. For six cDNA sequences of GhNAC1 to GhNAC6, each contained a single complete ORF encoding a predicted 276, 299, 298, 346, 356 and 327-amino acids protein with a calculated molecular mass of 31.9, 33.8, 33.9, 38.4, 40.2 and 37.0 KDa, respectively, as well as an isoelectric point of 5.83, 5.74, 6.54, 8.87, 8.17 and 5.39, respectively. All six have conserved intron-exon structure, although they differ in intron length and chromosomal location. The predicted proteins, GhNAC1- GhNAC6, are similar in sequence, especially in the NAC domain. Semi-quantitative RT-PCR reveals that all GhNAC genes were highly expressed in leaves while they had little to no expression in stems, roots and 7-day- post anthesis fibers. Based on real-time quantitative RT-PCR, the genes were differentially regulated under drought, high salt, cold and/or ABA conditions. Based on a phylogenetic analysis, six GhNAC belong to four NAC protein subfamilies, ATAF, AtNAC3, NAP and NAM. Three of these subfamilies, ATAF, AtNAC3 and NAP, include a large number of stress-regulated NAC genes in other plants. We identified an abiotic stress related bHLH-type transcription factor (GhbHLH1) for the first time in cotton. The phytohormone ABA was known to play a vital role in modulating plant responses to drought stress, and AtMYC2 (a GhbHLH1 homolog) had been proved to act as transcriptional activator involved in the dehydration and ABA response pathway in Arabidopsis. Here, using AtMYC2 amino acid sequence as a querying probe to screen Gossypium hirsutum L. EST database and contigging the candidate ESTs, the putative cDNA sequence of cotton bHLH transcription factor was assembled. Further a pair of specific primers flanking the sequence of open reading frame of cotton bHLH transcription factor was designed based on this assembled cDNA sequence, then a key gene GhbHLH1, positively regulating ABA response in cotton (Gossypium spp.) was isolated from leaves cDNA of drought-treated upland cotton cv Jinmian 19 by RT-PCR approach. The full-length cDNA of GhbHLH1 has an open reading frame of 2,025 bp, encoding a protein of 674 amino acids with a calculated molecular mass of 73.6 kDa and an isoelectric point of 5.46. Sequence alignment shows that GhbHLH1 contains a 60 amino acid long bHLH domain. We found that the deduced amino acid sequence of GhbHLH1 showed high homology with bHLH domain proteins in Arabidopsis, especially with AtMYC2, which plays an important role in response to stress stimuli. Semi-quantitative RT-PCR reveals that GhbHLH1 is strongly expressed in 7-day- post anthesis fibers but weak in roots, stems and leaves. Based on real-time quantitative RT-PCR, the expression of GhbHLH1 in leaves was transitorily induced by ABA and PEG treatments, although its transcripts were accumulated in various organs. However, its expression was not affected by salt and cold treatments. In addition, the further functional analysis of GhbHLH1 gene is in progress