【作者】 杨细燕；

【导师】 张献龙；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2009， 博士

【摘要】 棉花是重要的经济作物,也是重要的油料和蛋白质资源。棉花的生产关系着国计民生的发展。然而,各种各样的生物胁迫(病、虫害等)和非生物胁迫等因素严重制约了棉花生产。如今,棉花育种面临的主要问题是病虫害及其缺乏抗源。从棉花野生种中转移抗虫等重要农艺性状到栽培种中非常重要。尽管传统的育种方法对有益农艺性状的改良已经取得极大的进步,但是培育出突破性品种还是很困难。原生质体培养作为一项新的细胞工程技术,也越来越多的渗透到作物改良中,以其为基础的原生质体融合为克服有性杂交不亲和、转移有利性状、创造新的种质提供了一条有效途径。同时,在原生质体培养过程中,作为一种裸露的细胞,在合适的培养基上,原生质体能完成细胞壁再生过程,是研究细胞壁再生的良好材料。本研究主要内容涉及棉花原生质体培养和不对称融合及以棉花原生质体为材料通过抑制差减杂交法构建细胞壁建成的相关基因文库,并对部分基因进行功能验证,取得的主要结果如下:1.原生质体培养再生植株是原生质体融合中关键的一步,在开展棉花原生质体融合前,建立比较成熟的原生质体培养体系是基础。本研究在本实验成熟的陆地棉原生质体培养的基础上,以戴维逊氏棉为材料研究野生棉原生质体的培养条件。结果表明,在实验的7中激素组合中,0.45μM 2,4-D/2.68μM NAA/0.93μM kinetin对戴维逊氏棉原生质体培养最有效。原生质体密度在2-10×10~5 cells/ml比较适合原生质体分裂和愈伤形成;使用0.5 M葡萄糖或者0.1 M葡萄糖加0.5 M甘露醇能较好维持渗透压。在三种培养方法中,固液双层培养能达到较好的培养效果。2.在进行原生质体融合之前,往往采用不同的物理或化学因素钝化亲本一方的核或胞质,得到不对称融合杂种。本研究用不同剂量(0,38.7 J/cm~2,77.4 J/cm~2,116.1 J/cm~2)的紫外线照射克劳茨基棉的原生质体进行供体亲本细胞核失活的研究。琼脂糖凝胶电泳的结果显示,在对照(辐射剂量为0 J/cm~2)样品中DNA没有出现片段化,在辐射剂量为38.7 J/cm~2时,DNA出现少量片段化,片段较大,长度范围较窄,而辐射剂量为77.4 J/cm~2和116.1 J/cm~2时,DNA的片段化较严重,小片段数量增多。原生质体的活力随着辐射剂量的增加而逐渐降低,降低的差异比较显著。对于相同的处理剂量,随着培养时间的延长其活力也逐渐下降。所有经紫外线照射的原生质体到最后均未形成细胞团,而未经照射的原生质体能形成愈伤并最终再生植株。因此,我们选择38.7 J/cm~2为克劳茨基棉原生质体细胞核失活的最低辐射剂量。3.本研究中开展了多个组合的原生质体不对称融合。其中我们得到了以陆地棉(Coker201)为受体、野生棉(G.klotzschianum)为供体的组合(Coker201+G.klotzschianum)及以陆地棉鄂抗8号为受体、野生棉(G.stockii)为供体的组合(Ekang8+G.stockii)的不对称融合杂种,并对得到的杂种进行形态学、细胞学(流式细胞仪和染色体压片)及分子水平(RAPD、SSR、CAPS及cpSSR)的验证。大部分不对称融合杂种形态上偏向受体亲本,只有少数表现出双亲中间型,局部组织也有杂种优势表现。细胞学和细胞流式仪检测表明这些植株的核物质介于受体亲本及两亲本和之间,杂种染色体变化范围很大(2n=54-74)。RAPD和SSR分析再生植株的核基因组表明,亲本的DNA片段共存或缺失于不对称杂种中说明了再生植株的杂种真实性。对杂种和亲本胞质基因组的分析表明,不对称杂种的线粒体和叶绿体某些区段的DNA发生了交换。这是首次在棉花中实现不对称融合的报道。棉花原生质体融合的成功为棉花遗传育种提供了新的技术手段,也为利用不对称融合进行棉花的种植资源创新和棉花遗传改良提供了基础条件。实验得到的杂种植株将进行进一步研究分析,掌握融合杂种的核质基因重组规律,以期更好的应用于育种实践。4.植物的细胞壁结构复杂,由多种大分子聚合物组成。细胞壁合成的分子机理是现在分子生物学研究的热点问题。我们通过荧光增白剂对细胞壁的主要化学成分纤维素进行染色,结合显微观察,发现棉花原生质体在培养的前48小时内完成重建细胞壁。为了分离棉花原生质体重建细胞壁相关的基因,我们以棉花新鲜原生质体和培养后48小时内各时间点原生质体的mRNA为起始材料,通过抑制差减杂交法,获得了原生质体再生细胞壁过程中特异表达的cDNA片段。将这些差异表达的cDNA片段连入pGEM-Teasy克隆载体,建成原生质体再生细胞壁过程中的cDNA差减文库。通过差异筛选,得到了412个差异表达的克隆,进一步的测序和生物信息学分析得到了210个单一的ESTs。序列比对和表达谱分析表明这些ESTs可能在细胞壁合成中行使不同的生物学功能,有些ESTs的表达有时空特异性,包括一些已有报道的细胞壁合成相关基因家族的成员。RT-PCR和qRT-PCR结果也进一步证实了表达谱的结果。有些可能编码一些细胞壁蛋白的ESTs在细胞壁再生过程中优势表达,如脯氨酸丰富蛋白、甘氨酸丰富蛋白、伸展素、阿拉伯半乳糖蛋白及类膨胀素蛋白可能参与初生壁和次生壁的构建和修饰。蔗糖合成酶在细胞壁合成的早期有重要的作用。我们的实验中也分离了大量转录因子的ESTs,包括SPL14,锌指蛋白,NAC,IAA-re,MYB,WRKY,ATB2,RAV2,SMP1和RAD5等,对这些转录因子的进一步研究可能更深入的揭示细胞壁合成初期的表达调控机理。另外,我们的文库中还富集了一些Ca2+-CaM信号转导系统的因子,这也为研究Ca信号系统在细胞壁重建中的作用提供了依据。在对差减文库表达谱研究和一些目标ESTs深入研究的基础上,我们推测了一个可能的由原生质体再生细胞壁的可能的分子模型。5.在差减文库构建及RACE技术的基础上,我们克隆了四个类膨胀素基因(expansin-like),分离的两个基因分别命名为GhEXLA1、GhEXLA2、GhEXLB1和GhEXLB2。GhEXLA1全长cDNA为1236bp,ORF长度783bp,编码一个含260个氨基酸的蛋白质;GhEXLA2全长cDNA为961bp,ORF长度780bp,编码一个259个氨基酸含蛋白质;GhEXLB1全长cDNA为1028bp,ORF长度780bp,编码一个含259个氨基酸的蛋白质;GhEXLB2全长cDNA为1172bp,ORF(771bp),一个含255个氨基酸的蛋白质。基因结构研究发现GhEXLA1、GhEXLB1和GhEXLB2都具有三个内含子,GhEXLA2具有四个内含子。同源性分析显示GhEXLA1和GhEXLA2编码的蛋白质都与栎树的expansin-like protein(CAE12163)同源性最高,GhEXLB1编码的蛋白质与毛果杨的假想蛋白(PtrEXLB1)同源性最高,GhEXLB2编码的蛋白质与葡萄的假想蛋白(LOC100262694)同源性最高。对四个基因编码的蛋白序列进行生物信息学分析,发现它们都含有氨基端的信号肽和结合多糖的结构域,羧基末端含有类似结合纤维素的结构域。此外,我们还利用RNAi和超表达来研究这两个在细胞壁发育过程中的准确功能。我们已经分别构建了几个基因的RNAi载体和GFP融合表达载体,已经转化棉花,并获得了两个基因GhEXLA1和GhEXLB1的RNAi遗传转化再生植株;同时GFP融合表达载体转化拟南芥,获得了T2代植株,转化植株的基因表达分析正在进行中。更多还原

【Abstract】 Cotton is an important economic crop,is also important resources of oil and protein. Its production directly related to people’s livelihood.But the unit yield increase has been limited by numerous diseases and pests that cause serious yield reduction.Now,the main problems are related to pests,diseases,and their control.The transfer of polygenic traits such as pathogen resistance from wild species to cultivars is of great importance in cotton. Although conventional breeding programs have made steady improvements in agronomic traits,it is becoming more and more difficult to develop new varieties.Protoplast culture emerging as a new cell engineering technology has take part in crop improvement.On this base,an efficient way to bypass sexual-crossing barriers and create new germplasm in cotton was developed by protoplast fusion through which we can transfer desirable agronomical relevant traits from wild cotton to cultivars.At the same time,protoplasts retaining the capacity to develop a normal cell wall under suitable culture conditions were regarded as particularly suitable for studying cell wall biosynthesis.Our studies involved protoplast culture,asymmetric protoplast fusion between cultivars and wild species and identification of genes associated with cell wall biosynthesis by suppression subtractive hybridization based on cotton protoplast.The main results of this research were as fellows:1.An effective protoplast culture system is necessary for obtaining somatic hybrids via cell fusion.In this study,Protoplast was isolated from wild cotton（Gossypium davidsonii） was cultured in KM₈P medium supplemented with different phytohormones. The most effective combination was 0.45μM 2,4-dichlorophenoxyacetic acid,2.68μMα-naphthalene acetic acid and 0.93μM kinetin and the division percentage at the 8^th day was 30.78±3.04%.The density of protoplast at 2 - 10×10⁵ cells/ml was suitable for protoplast division and calli formation,with a division percentage of 32.21±3.64%and a plating efficiency of 9.12±2.61%at the 40^th day.The optimal osmotic potential was achieved using 0.5 M glucose or 0.1 M glucose plus 0.5 M mannitol.Protoplasts were cultured in three ways,a double-layer culture system,with liquid over solid medium was proved to be the best way.2.For transferring partial genome or cytoplasm,the parent protoplasts were usually treated with chemical or physical methods prior to fusion.Ultraviolet（UV） irradiation was regarded as a substitute or alternative to ionizing irradiation in asymmetric protoplast fusion experiments.In this paper,we conducted four doses（0,38.7 J/cm²,77.4 J/cm², 116.1 J/cm²） of UV irradiation on G.klozschianum protoplasts.The results of agarose gel electrophoresis indicated that considerable fragmentations of the DNA happened when irradiated with the dose of 77.4 J/cm² and 116.1 J/cm²,but less fragment action with the dose of 38.7 J/cm² compared with control.The viability and first division percentage of the irradiated protoplasts decreased along with the increase of irradiation dose,and the viability of irradiated protoplasts decreased along with the increase of culture time.All the protoplasts treated with UV could not form mass callus although plantlets developed from untreated protoplasts.As a result,and the dose of 38.7 J/cm² UV treatment was chosen as the lethal dose for the following asymmetric protoplast fusion experiments.3.Asymmetric protoplast fusion was carried out between some combinations. Hybrid plants were obtained between two combinations Coker 201（as receptor） + G. klotzschianum（as donor） and EKang 8（as receptor） + G.stockii（as donor）,and the hybrids were analyzed at morphological,cytological（chromosome counting and flow cytometric analysis） and molecular levels（RAPD、SSR、CAPS and cpSSR）.Most regenerated plants derived from fused protoplasts displayed a recipient-like morphology, while some showed an intermediate phenotype.And some tissues showed heterosis. Chromosome numbers in these somatic hybrids displayed a large range.Cytology and cell flow cytometry showed that the nuclear material of these plants were between the receptor and the sum of the two parents.Absence or co-existence of parents’ genome DNA fragments was identified through molecular analysis.The heredity of cytoplasm was investigated by cleaved amplified polymorphic sequence（CAPS） analysis and cpSSR. The results indicated that recombination and rearrangements might have occurred in some regions of mitochondria（mt） and chloroplast（cp） DNA.To our knowledge,this is the first report about asymmetric protoplast fusion in cotton.Protoplast fusion has provided a new way for cotton genetics and breeding,as well as the basis for creation cotton genetic resources,and the hybrids obtained would be useful for breeding programs.4.The plant cell wall is of super molecular architecture,and is composed of various types of heterogeneous polymers.The genes expression during cell wall biosynthesis attracts much attention for biologist.However,the molecular mechanisms underlying cell wall biosynthesis are currently poorly understood.Microscopic analysis,using Calcofluor White to stain cellulose,showed that the protoplasts generated a new cell wall in the first 48 h after transfer to a wall-regeneration medium.To identify genes related to cell wall biosynthesis in cotton,suppression subtractive hybridization was used to visualize differential gene expression at seven distinct time-points within these first 48 h.Around 412 differentially expressed ESTs（＞3 fold） were identified,and 210 unigenes were sequenced successfully.As confirmed by real-time RCR and quantitative RT-PCR analysis,the selected genes displayed complex expression pattern during cell wall regeneration in protoplast,including most previously published cell wall associated genes. Some ESTs similar to cell wall protein genes such as proline-rich protein（PRPL）, glycine-rich protein（GRP）,extension（EPR1） and fasciclin-like arabinogalactan protein （FLA2） which might participate in primary cell wall or secondary cell wall construction and modification were up-regulated during cell wall regeneration in cotton protoplast. Sucrose synthase,an important enzyme in sugar signal pathway,played important roles in cell wall regeneration for cellulose biosynthesis.This study highlighted the function of some transcription factors on cell wall biosynthesis,including SPL14,NAC,Gbiaa-re, MYB,WRKY,SMP1,RAD5 and zinc finger family protein.Enrichment of Ca2+-CaM signal molecules in cell wall biosynthesis was also displayed.On the basis of expression profiles of genes involved in cell wall regeneration during protoplast culture,we proposed a model of cell wall regeneration from protoplast derived of cotton suspension cultures.5.Based on the cell wall regeneration SSH library screen and Gene-RACE strategies, four genes encoding plant cell wall related expansin-like proteins have been isolated from upland cotton,named GhEXLA1,GhEXLA2,GhEXLB1 and GhEXLB2 respectively. GhEXLA1 was 971bp in length,and had an open reading frame（ORF） of 783bp which encoded a predicted polypeptide of 260 amino acids（aa）.GhEXLA2 was 961bp in length, and contained an ORF of 780bp which encoded a predicted polypeptide of 259 aa. GhEXLB1 was 1028bp in length,and contained an ORF of 780bp which encoded a predicted polypeptide of 259 aa.GhEXLB2 was 1172bp in length,and contained an ORF of 771bp which encoded a predicted polypeptide of 255 aa.Gene structure analysis showed that GhEXLA1,GhEXLB1 and GhEXLB2 have 3 introns,but GhEXLA2 have 4 introns.Homologic searching against protein database using tBLASTx in GenBank,the deduced protein sequences of GhEXLA1 and GhEXLA2 were found to share the highest protein homology with robur expansin-like protein（CAE12163）,GhEXLB1 with Populus trichocarpa hypothetical protein（PtrEXLB1）,and GhEXLB2 with Vitis vinifera hypothetical protein（LOC100262694）.The bioinformatics analysis of their proteins revealed they owned an N-terminal signal sequence and EG45-like domain,C-terminal cellulose-binding-like domain.Furthermore we verify the precise functions of the genes on cell wall regeneration by RNA interference and GFP-fusion expression approach.We have constructed the RNA interference（RNAi） and GFP-fusion expression vector.These vectors were transformed into upland cotton mediated by Agrobacterium,and we have obtained RNAi transgenic plants of GhEXLA1 and GhEXLB1.At the same time,the GFP-fusion vectors have transformed into Arabidopisis,and T2 transgenic plants were obtained.The analysis of regeneration plants are on going.更多还原