【作者】 张海平；

【导师】 王学德；

【作者基本信息】 浙江大学 ， 作物遗传育种， 2008， 博士

【摘要】 棉花黄萎病是棉花生长过程中一种最具破坏力的真菌病害，在世界范围内流行。该病害不仅严重影响棉纤维产量，降低幅度达20-60％，而且能大幅度降低棉纤维的品质。棉花黄萎病已经成为世界上棉花生产的主要障碍。利用遗传工程技术改良棉花品种可以提高棉花的抗病性。农杆菌介导法是目前应用最广泛、转化机理最清楚的基因转化方法。然而，农杆菌介导的棉花基因转化存在很多问题，如棉花组织培养的基因型依赖性、转化效率低、转基因植株基因表达沉默等等，这些问题已经成为棉花基因工程研究领域的障碍和瓶颈。本研究主要以陆地棉品系5983为材料，优化了组织培养体系和基因转化体系，将从苜蓿种子中克隆的苜蓿抗菌肽基因导入到棉花中，经检测获得了抗黄萎病的转基因棉花。棉花纤维的生长发育是依赖于一系列基因的调控。但由于棉花基因遗传转化需要大量的人力和物力，转化和生长周期长，大规模进行棉纤维基因功能分析非常困难，基因是如何调控棉纤维的生长发育还不是很清楚。因此，有必要建立一套快速简单的棉纤维基因功能鉴定的瞬时表达方法。本研究以GUS为报告基因，采用棉花胚珠离体培养技术，基因枪转化、子房注射法，建立了两套外源基因转化棉纤维的瞬时表达体系。将取得的研究成果归纳如下：1．棉花组织培养体系的优化以5-7日苗龄无菌苗下胚轴切段为外植体，在以LS和MSB培养基为基本培养基，共15种不同激素配比培养基上诱导愈伤组织。结果表明，不同培养基类型和激素组合对愈伤组织的诱导都有重要的影响。同样激素处理条件下，LS培养基上诱导出的愈伤组织要好于MSB培养基，愈伤组织大都为黄绿色，具有分化为胚性愈伤组织的潜力。在所有的激素组合中，在含0．1mg／L KT和0．2mg／L2，4-D的LS培养基中诱导的愈伤组织最好，颜色为黄绿色，质地松软。而其他组合的培养基上诱导的愈伤组织则呈白色硬块或褐色稀泥状，分化为胚性愈伤组织的潜力小。将获得的愈伤组织继代到四种不同的培养基上，在MSB培养基上增殖的愈伤组织的色泽和质地要好于LS培养基上的愈伤组织。在MSB培养基上增殖的大部分愈伤组织为淡绿色或粉红色，而LS培养基上增殖的愈伤组织则是白色硬块愈伤。ZT或活性炭的加入降低了愈伤组织的增殖速度。将在MSB培养基上增殖的愈伤组织转移到6种含不同激素种类及配比的MSB培养基上。结果表明，0．3mg／L KT和0．5mg／L IBA组合的效果优于其他组合，在这个培养基上产生的子叶胚数和总胚数最多。获得的胚状体放在含0．5 mg／L NAA的1／2MSB培养基上诱导绿苗形成。5μmol/LNi²⁺和Zn²⁺能促进胚性愈伤组织的分化和绿苗的萌发。待幼苗长到5-8cm时，采用直接移栽和嫁接移栽的方法将苗移到温室。2．棉花转基因体系的优化以含质粒pCAMBIA1301的农杆菌介导棉花下胚轴切段，50mg／L的潮霉素加入到培养基中筛选抗性愈伤组织，优化了农杆菌介导的棉花遗传转化的影响因子。研究结果表明：不同农杆菌菌种对棉花的浸染能力不同，在三种农杆菌菌种中，农杆菌LBA4404的浸染能力最强。农杆菌菌液浓度OD₆₀₀值在O．5-0．7时，培养基中加200μmol／L乙酰丁香酮，浸染10-15min，在50mg／L潮霉素培养基筛选后，愈伤组织转化率最高。共培养温度和时间对愈伤组织转化率也有明显的影响。研究表明在共培养温度21℃下，共培养60 h，转化效果最好。3．植物表达载体的构建与转基因棉花的筛选从苜蓿种子中克隆了已知基因苜蓿抗菌肽基因（alfAFP），并将其连接到质粒pCAMBIA1301中。基因的表达由CaMV35S启动子驱动。用已经优化的基因转化体系进行目的基因转化，待再生苗5-8 cm高时，将其直接移栽到温室，最后共得到15颗再生植株。经潮霉素涂抹，发现有3株是假阳性植株。对其余12个转基因植株进行分子检测，PCR结果表明在12个转基因植株的基因组DNA中都有目的基因存在，而在对照植株的基因组DNA中检测不到目的基因的存在。随后，为检测外源基因在棉花基因组中的整合，对这12个转化植株进行Southern检测。结果表明，在12个转化植株中都能检测到Southern杂交信号，有6株是单拷贝整合，另外6株是双拷贝整合。提取12个转基因植株根部的RNA，Northem检测都有杂交信号，说明外源DNA在转基因植株根部都有转录。4．转基因棉花的抗病性鉴定、生理特性表现和农艺性状表现离体抗病检测结果表明，转化植株叶片粗提液的处理产生的菌落数显著（P＜0．01）少于对照，大丽轮枝菌的生长被抑制46．2-86．8％，表明外源抗菌肽在转基因棉叶中含量较高。为鉴定转基因植株的抗病性，用50ml大丽轮枝菌孢子悬浮液接种转基因植株和对照植株的根部。在成熟期调查黄萎病的发病级别。结果表明，12个转基因植株中，有5株是黄萎病免疫株，有3株为1级发病，3株是2级发病，有1株发病严重，是3级。离体抗病检测和转基因植株接菌检测结果都表明，alfAFP基因的导入不同程度的提高了棉花对黄萎病的抗性。抗病鉴定的同时，我们测定了四个与抗病相关的酶在接菌后的活性变化。四个酶活性的变化趋势相同。对照植株接菌后酶活性有一个快速升高和快速降低的过程。转基因植株中，部分抗性植株酶活性变化也有一个升高和降低的过程，但变化幅度较小，而黄萎病免疫植株的酶活性变化则相当平缓，没有明显的峰值。外源基因的导入对棉花农艺性状也有较大的影响。转基因植株株高普遍降低，果枝数、果节数和铃数显著减少，铃重大大降低，但对衣分的影响不大。在纤维品质性状中，纤维比强度下降，马克隆值有不同程度的增加，纤维长度的变化不一致，有的增长，有的变短。5．棉纤维基因转化的瞬时表达体系的建立1DPA胚珠预培养1 d后，用基因枪轰击。在轰击压力1100psi，射程9 cm，真空度28英寸汞柱，质粒用量为2μg/枪的条件下，GUS染色瞬时表达效果最好。轰击后GUS染色表明，该基因可以在纤维发育的第3-22天持续表达，即该转化体系可以检测纤维发育第3-22天内相关基因的表达。子房注射法转化外源基因，质粒浓度为0．04-0．07μg/μl之间时转化效果最好。注射开花后1DPA子房，胚珠GUS染色表明，注射后第3天的GUS表达率最高，外源基因可以在纤维中持续表达9天。即该转化体系可以检测纤维发育第2-10天内相关基因的表达。更多还原

【Abstract】 Verticillium wilt,caused by Verticillium dahliae（V.dahliae）,is one of fungal diseasedecreasing not only fiber yield by 20-60% but also fiber quality,and it exist widely inworldwide cotton planting area.Now,it has been one of major limiting factors in cottonproduction.Results from previous researchers showed that cotton varieties could be improvedin resistance of disease,pest and herbicide and fiber quality etc by using gene engineering.Inrecent years,the gene transformation via Agrobacterium-mediated system was used widelyand its mechanism of transformation is very clear.However,many problems still exist incotton gene transformation.The main reasons are a higher degree of genotype dependence inplant regeneration of cotton tissue culture,lower efficiency in gene transformation of cottonand foreign gene silence in transgenic plant.In this paper,an optimized system wasestablished for cotton tissue culture and gene transformation.An Alfalfa antifungal peptide（AlfAFP）gene,cloned from seeds ofMedicago sative,was introduced into cotton genome andtransgenic plants of enhanced resistance to V.dahliae were obtained.Development of cotton fiber was regulated by many genes.However,because of longduration and a lot of work in cotton gene transformation,large-scale analysis of gene functionin fiber development is very difficult by using cotton stable transformation system.Therefore,it is not clear that which and how genes regulate fiber development.It is very necessary toestablish a gene transient expression system to evaluate gene function in fiber cell by asimpler and quicker test method.In this paper,two transient gene expression systems in fibercell were established by particle bombarding ovules and injecting ovaries through pollen tubepathway.The main results are listed below:1.Optimization of effect factors in cotton tissue cultureCotton hypocotyl pieces（3-5 mm）from 5 to 7 days-old sterile seedlings were culturedon 15 kinds of callus induction media,which included different kinds of hormones in LS orMSB basic media.Results indicated that calli growth on LS media were better than these onMSB media when hormones and hormone concentrations were same.Most of calli on LSmedia were yellow-green and had capacity to differentiate into embryonic calli.In all hormone treats,calli growth on LS medium with 0.1 mg/L KT and 0.2 mg/L 2,4-D were thebest,and the calli were yellow-green and loose.Calli on the other media were white and hardor brown and very soft,which were not impossible to differentiate into embryonic calli.Yellow-green calli were placed on 4 proliferation media and showed a better proliferation onMSB medium than on LS medium.Proliferation efficiencies were decreased when ZT oractive carbon were added in MSB medium.When calli on MSB medium were transferred on6 kinds of media with different hormones,the MSB medium with 0.3mg/L KT and 0.5 mg/LIBA was the best and could produced the highest number of embryo and cotyledon embryo inall treats.If somatic embryos were developed into plantlets on 1/2MSB medium with 0.5mg/LNAA,differentiation efficiency of embryogenic calli and plantlet regeneration was increasedwhen 5μmol/L Zn²⁺and Ni²⁺were added in medium.It was suggested that plantlets withroots should be transplanted into greenhouse when plants heights were about 5-8 cm andSeedlings without roots were grafted on rootstock in greenhouse.2.Optimization of effect factors in cotton gene transformationEffect factors of transformation processes were examined in efforts to improve theproduction efficiency of transgenic cotton.The result showed that strain LBA4404 wassignificantly better than EHα101 and EHα105.The efficiency of transformation could beimproved when bacterium density was 0.5-0.7 at OD₆₀₀.15 minutes is the most appropriatetime for cotton hypocotyl pieces to be dipped into Agrobacterium suspension.Relative lowco-cultivation temperature（21℃）and long co-cultivation duration（60h）were optimal fordeveloping a highly efficient method of cotton transformation.Concentration ofacetosyringone at 200μmol/L during co-cultivation significantly increased transformationeffiency.3.Construction of plant expression vector and development of transgenie plantsAn alfalfa antifungal peptide gene（alfAFP）,which was cloned from seeds of Medicagosative,was ligated in pCAMBIA1301 to form a recombinant pCAMBIA1301-alf.The alfAFPgene expression was under the CaMV35S promoter control in recombinant binary vector.Gene alfAFP was delivered into an upland cotton line 5983 via Agrobacterium-mediatedhypocotyl system.It took about 8-10 months that 15 plants were regenerated by cultivatingthe transformed cotton tissues.Transgenic plants were transplanted in greenhouse when their roots were about 5cm in length.50mg/L hygromycin solution.was spread onto the yongleaves oftransgenic plants.7-10 days later,results showed that there were 3 negetive plants in15 transgenic plants.PCR products confirmed the integration of the alfAFP gene in thegenome of 12 transformants.Southern blot analysis showed that 6 transformants were onecopy and another 6 transformants were two copies of the alfAFP gene.RNA from roots oftransformants was used for Northern blot and gene transcripts were present in all 12transgenic plants.4.Resistance to V.dahliae,changes of related enzymes and agronomy characters oftransformantsIn vitro assays showed that crude leaf protein extracts from transforrnants was able tosignificantly（p＜0.01）inhibit the growth and proliferation of V.dahliae by 46.2-86.8%compared to extracts from control plants.In vivo assays with fungal pathogen showed thattransformants displayed a significant reduction in disease symptom compared to controlplants.At harvest stage,evaluation of disease degree demonstrated that 5 plants of 12transformants were immune to verticillium wilt,3 plants each showed the disease grade of 1,another 3 plants were scored 2,respectively.1 plant showed severe disease symptom with thedisease grade of 3.Results from assays in vitro and in vivo suggested that the alfAFP genewas able to express in the upland cotton and its product of gene expression was active to thegrowth inhibition of V.dahliae.Activities of four related enzymes in control and transgenic cottons were measured afterinoculated by V.dahliae suspension.Results showed that activity trends of four relatedenzymes were similar.After inoculated,enzyme activities of control plants dramaticallyincreased,and then decreased rapidly.There were peaks at 3 or 5 d after inoculation.Intransgenic plants,changes of enzyme activity of transgenic plants with part verticillium wiltsymptom were similar to that of control plants,but had lower peaks.However,the enzymeactivity of immune-verticillium wilt cotton was maintained at low level and did not show anyobvious peak.Agronomy characters of transgenic cotton plants were influenced by the alfAFPintroduction.Results showed that transgenic plants were reduced in height,fruiting branchesand nodes,bolls and boll weight,but was not affected in fiber lint percentage.In characters of fiber quality,transgenic plants showed lower fiber strength and higher micronaire value,buthad significantly unstable change in fiber length,some became longer and others becameshorter than control plants.5.Establishment of transient expression system of foreign gene in cotton fiber cell1DPA ovules were cultivated for 1 d on BT medium,and then they were used for thebolistic transformation.The optimal parameters were as follows:Helium pressure at 1100psi,target distance at 9 cm,the vaccum pressure at 28 inches Hg,vector concentration at 2μg perbombardment.Foreign gene expression could be continually detected in fiber cells onculturing ovules from 2 to 20 d after bombardment.After 20 d,gene expression disappeared.It is indicated that function analysis of genes lelated with the fiber developmet may beavailable by the bolistic transformation system when ovules were cultured in the period from3 to 22d in fiber development.Vector concentration of 0.4-0.7μg/μl was the optimum for gene transformation for 1DPA ovules by the method of ovary-injection in vivo.GUS gene in fiber cells on ovules couldcontinually express within 9 days after transformation,and its expression efficiency was themost high at 3 d after injection.Therefore,the duration from 2 to 10 d may be the best timefor function evaluation of genes related with fiber development by the ovary-injection system.更多还原