摘要
河北杨(Populus hopeiensis Hu et Chow)属杨柳科(Salicacae)杨属(Populus L)白杨派(Section Leuce Duby)树种,是一个主要分布在我国西北与华北干旱半干旱地区的重要乡土杨树,它抗旱、抗寒、耐瘠薄、根蘖性强,是进行干旱胁迫相关基因克隆与研究林木干旱胁迫信号转导与耐受性机理的理想材料。本研究以河北杨为试验材料,建立了其组织培养无菌体系,利用cDNA-AFLP差异表达技术对河北杨干旱胁迫响应基因进行筛选,获得了457条TDFs片段。在对其进行分析的基础上,通过同源克隆结合RACE方法从干旱胁迫处理的河北杨中分离了植物CBF/DREB1与DREB2类转录因子基因:PhCBF4a/4b、PhDREB2a/2b,进而对其序列及组织特异性表达模式与不同胁迫条件下的表达模式进行了系统的分析。同时,通过瞬时表达研究与转化拟南芥研究,进一步鉴定了PhCBF4a/4b、PhDREB2a/2b基因的功能。经上述研究主要得到如下结果:
1.以干旱处理不同程度的河北杨无菌生根苗为试验材料,使用256对选择性扩增引物对河北杨干旱胁迫响应基因进行了较为全面的筛选,共回收了484条TDFs片段,最后测序获得457条片段序列。其中,391条TDFs在NCBI蛋白数据库中有同源基因,其余38个在杨树EST数据库中有同源序列,25个在杨树基因组数据库有类似序列,而TDF45-1、TDF183、TDF280-1在三个数据库中均没有类似序列。根据条带强度变化模式,对TDFs条带进行了分级聚类,共分为12个类群,其中Cluster8与Cluster9分别包含118与106条TDFs片段,其表达模式为瞬时上调然后下降或持续上调。这两个类群的TDFs数目占所有片段的49.0%,暗示了这两类基因在河北杨干旱胁迫响应中的重要作用。在所获得的TDFs片段中,有些TDFs片段代表了同一个基因或同一个基因家族的不同成员,也就是说,在干旱胁迫诱导的河北杨中,这些基因的转录本数较多,显示了这些基因在调控河北杨干旱胁迫耐受性中的重要作用。
2.通过同源分析方法,推测杨树CRT/DRE顺式作用元件的核心基序为A/GCCGACA/G,不完全等同于拟南芥CRT/DRE的基序A/GCCGACNT。利用这一推测模式,发现在获得的457条TDFs片段中有8条可能是河北杨DREB转录因子的靶基因。其中有4条是LEA或ERD类的基因,另外4条可能参与了基因转录或翻译过程,而在拟南芥、水稻与玉米中,第二类的基因并没有占如此大的比例,这一结果显示虽然DREB调控子在植物中普遍存在,但并不完全一致。
3.利用同源克隆结合RACE方法从干旱处理的河北杨中克隆了两对DREB类基因:PhCBF4a/4b、PhDREB2a/2b。虽然存在Indel区段与氨基酸残基的替换,但在氨基酸水平上,其序列一致性分别达到94.0%与97.7%,而且其同源基因在其它杨树及杨树杂交群体中的存在模式并不一致,推测它们可能分别是一对等位基因。同时,利用半定量PCR方法对其组织特异性表达模式与不同胁迫条件下的表达模式进行了分析,发现PhCBF4a/4b主要受干旱、低温与NaCl胁迫的诱导,而PhDREB2a/2b基因受干旱、高温与NaCl胁迫条件诱导而较强表达;在各种胁迫条件下,PhDREB2a/2b的表达量基本上均高于PhCBF4a/4b,而且在正常条件下,PhDREB2a/2b也能检测到表达;同时,在各种胁迫条件下,PhCBF4a与4b、PhDREB2a与2b表达模式也并不完全一致。这些结果暗示了PhCBF4a与PhCBF4b、PhDREB2a与PhDREB2b在调控河北杨胁迫抗性中的不同作用。
4.对PhCBF4a/4b同源基因进行的进化发育分析显示,植物CBF/DREB1类基因经历了复杂的进化事件。在从共同的双子叶祖先分离之前或分离过程中,CBF/DREB1基因经历了多次的复制与趋异事件。在双子叶亚家族中,CBF/DREB1基因的分类部分的平行于物种分类,表明部分CBF基因是在物种形成之后才开始进行复制与趋异的进化过程。
5.瞬时表达研究发现PhCBF4a/4b、PhDREB2a/2b具有明显的转录激活功能,而且PhCBF4a与4b、PhDREB2a与2b的转录激活功能有显著的差异,说明其序列的差异导致了其功能的差异,并可能影响到其在河北杨干旱胁迫耐受性中的功能。
6.通过拟南芥遗传转化研究,发现与对照拟南芥植株相比,转PhCBF4a/4b、PhDREB2a/2b基因拟南芥生长迟缓、开花期延迟,在正常条件下,其DREB靶基因的表达量明显提高,干旱胁迫耐受性也明显提高,进一步证实了PhCBF4a/4b、PhDREB2a/2b的转录激活功能及其在调控河北杨胁迫响应中的重要作用。
7.通过重叠PCR方法删除了PhDREB2a/2b 3'编码区的两个PEST序列,瞬时表达研究与转化拟南芥研究证实获得的PhDREB2bP12d基因的转录激活功能并没有提高,初步说明河北杨PhDREB2a/2b可能不存在类似于拟南芥AtDREB2A的翻译后调控过程。
总之,通过研究初步筛选获得了河北杨干旱胁迫响应的TDFs片段,同时分离了河北杨DREB类基因并对它们进行了进一步的分析与功能鉴定,为河北杨及其它杨树干旱胁迫相关基因与干旱胁迫耐受性机理的进一步研究及林木抗旱遗传改良奠定了基础。
Populus hopeiensis Hu et Chow is a native species in Section Leuce,which is mainly distributed in northwest of China and north China.It's a drought- and chilling-tolerant tree species,therefore being an ideal material to study the mechanism of drought tolerance of woody plants.However,previous studies in P.hopeiensis mainly focused on in vitro culture,screening of somatic stress-resistant mutants,hybridization,and so on.In present study,drought stress responsive genes were screened with cDNA-AFLP in P.hopeiensis. Meanwhile,two pairs of highly similar DREB-like genes,PhCBF4a/4a and PhDREB2a/2b, were isolated from drought-stressed in vitro cultured plantlets,and their tissue-specific expression pattern and expression profile under various stress conditions were also studied. In addition,by transient expression assay and transgenic Arabidopsis assay,their function differences were characterized.The major results and conclusions are described as follows:
1.Drought stress responsive genes were screened with cDNA-AFLP,and 484 differential expressed TDFs were excised from PAGE gels.457 TDFs were successfully cloned,among which,454 TDFs were found homologs in NCBI protein Database,or in Populus EST Database,or in Genome Database of P.tricocarpa,but the rest 3 TDFs, TDF45-1、TDF183、TDF280-1,found no homologs,meaning that they are probably specific genes in P.hopeiensis.Based on the expression patterns of TDFs in the different drought-stressed plantlets,the TDFs were classified into 12 clusters.And the Cluster8 and Cluster9 contained 118 and 106 TDFs respectively,occupying 49.0%of all obtained TDFs, suggesting their important roles in response to drought stress in P.hopeiensis.Meanwhile, some genes or gene families were represented by more than one TDF,also suggesting their roles in regulation of drought stress tolerance in P.hopeiensis.
2.Using homolog analysis,the putative core motif of CRT/DRE cis-acting element was identified as A/GCCGACA/G in Populus.Corresponding to this motif,8 of the cloned 457 TDFs were presumable target genes of DREB-related genes of P.hopeiensis.Among them,4 TDFs shared high identity with LEA or ERD genes in other plants,and the rest 4 TDFs were probably involved in gene transcription or translation.
3.By RT-PCR and RACE-PCR,PhCBF4a/4b and PhDREB2a/2b were isolated from drought stressed plantlets in P.hopeiensis.Although indel fragment and substitution of amino acids residue were found in their coding region,PhCBF4a and PhCBF4b, PhDREB2a and PhDREB2b,shared a 94.0%and 97.7%overall amino acid sequence identity each other respectively,suggesting that PhCBF4a and PhCBF4a,PhDREB2a and PhDREB2b,were two pairs of alleles,i.e.,in terms with PhCBF4a/4b and PhDREB2a/2b, P.hopeiensis was heterozygous.It was further characterized by studying the occurrence patterns of their orthologous genes in other Populus or cross population.In addition,the expression profiles of PhCBF4a/4b and PhDREB2a/2b were characterized with semi-quantitative PCR.Expression of PhCBF4a/4b was induced by drought,cold and NaCl stresses,whereas drought,heat and NaCl stresses upregulated expression of PhDREB2a/2b.Meanwhile,the expression amounts of PhDREB2a/2b were higher than those of PhCBF4a/4b under stress conditions.Moreover,PhCBF4a and PhCBF4b, PhDREB2a and PhDREB2b,showed similar but not identical expression patterns.All these revealed their similar but not identical roles in response to stresses in P.hopeiensis.
4.A comprehensive phylogenic analysis revealed that the PhCBF4a/4b-like genes underwent complex evolution events in plants.In the eudicot subfamily,the existence of multiple subclades,each of which includes CBF gene members from Arabidopsis and P. tricocarpa,suggested that CBF genes were duplicated multiple times and diverged before or during speciation from the common eudicot ancestor.Meanwhile,clustering of the CBF homologs partly parallels the taxonomic classification,suggesting that duplication and divergence of some CBF genes occurred after speciation.
5.Characterization of trans-activation of PhCBF4a/4b and PhDREB2a/2b was performed with transient expression assay.The results showed that PhCBF4a/4b and PhDREB2a/2b could activate expression of GUS gene,thus were trans-active.At the same time,significant differences of trans-activity of PhCBF4a and PhCBF4a,PhDREB2a and PhDREB2b were found,confirming that the indel and substitutions of amino acid resulted in the different trans-activity,thereby presumably affected their functions in regulating responsiveness to stresses in P.hopeiensis.
6.Compared with the Control plants,overexpression of PhCBF4a/4b,PhDREB2a/2b in Arabidopsis not only induced strong expression of DREB target genes under unstressed conditions but also caused dwarfed phenotypes,later flowering in the transgenic plants.In addition,the transgenic plants also revealed elevated drought tolerance.All these also confirmed the trans-activation of PhCBF4a/4b and PhDREB2a/2b.
7.With over-lapping PCR,two PEST fragments in 3' coding region of PhDREB2a/2b were deleted.However,the subsequent transient expression assay and transgenic Arabidopsis assay revealed that trans-activity of the obtained PhDREB2bP12d wasn't promoted.On the contrary,its trans-activity was reduced,so it could be preliminarily concluded that the post-translation regulation in AtDREB2A doesn't exist in P.hopeiensis.
Taken together,the obtained results and conclusions gave new insights not only into elucidating the molecular mechanism of drought tolerance in P.hopeiensis but also into the genetic improvement of drought tolerance of woody plants.
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