【作者】 张高阳；

【导师】 祁建民；

【作者基本信息】 福建农林大学 ， 作物遗传育种， 2014， 博士

【摘要】 黄麻(Corchorus capsularis L.),又称绿麻,为椴树科黄麻属一年生草本韧皮纤维作物,是世界上最重要的长纤维作物之一,产量和种植面积仅次于棉花。由于黄麻纤维产量高、有光泽、吸湿性能好、散水快、易降解、质地柔软的特点,在商业上有金色纤维之称。近年来随着育种家的不断努力,黄麻纤维产量有了大幅度的提高,但由于黄麻中纤维素总体含量较低和木质素含量较高,导致黄麻韧皮纤维细胞壁木质化、单纤维细胞较短、粗硬、弹性小、可纺支数低等,严重影响黄麻纤维在面料纺织品上的有效应用和经济效益。植物组织培养是基因工程操作的重要基础,在大部分植物中,愈伤组织是实现农杆菌介导遗传转化的直接受体,同时也是获取原生质体、体细胞杂交、植株再生等的重要基础。目前有关黄麻组织培养和再生体系的研究较少,这严重制约着黄麻基因工程的发展。本研究主要研究内容为：利用不同公司的RNA提取试剂盒和和提取方法,优化了高质量黄麻RNA提取方法；利用优化的快速获取基因5’端的技术体系,获得了黄麻纤维素合成相关基因UGPase、CesAl和CCoAOMT基因cDNA片段并对该基因进行了功能鉴定；利用不同培养基和激素配比,研究了黄麻组织培养再生体系；利用韧皮部特异启动子定向沉默黄麻CCoAOMT基因。最后利用所获得的UGPase基因转化黄麻,来研究利用UGPase和CCoAOMT基因改良黄麻品质的可行性,主要研究结果如下：1.1黄麻组织培养及遗传转化体系的优化以圆果种黄麻“黄麻179”为材料,确定了以子叶和幼茎为外植体产生愈伤组织最佳培养基分别为：MS培养基中添加0.5mg/L TDZ+0.1mg/L或0.5mg/LIAA,MS培养基中添加2.0mg/L6-BA+0.5mg/L IAA和MS培养基中添加0.25mg/L TDZ+0.1mg/L或0.5mg/L NAA, MS培养基中添加0.5mg/L6-BA+0.5mg/L NAA。子叶愈伤组织再分化培养基为MS+0.5mg/L6-BA+0.5mg/LNAA,幼茎愈伤组织再分化培养基为MS+0.5mg/L TDZ+2.0mg/L IAA,通过筛选不同种类的褐变抑制剂,最终确定以0.1%的植酸作为黄麻组培中褐变抑制剂效果较好,不定芽诱导中,最终确定子叶节不定芽诱导最佳培养基是MS+2.0mg/L6-BA+0.25mg/L NAA+100mg/L HC。诱导率达到36.4%,不定芽或苗根诱导最佳培养基为MS+0.5mg/L6-BA+1mg/L NAA。选用200mg/L Cef作为子叶节遗传转化的抑制农杆菌生长的抑制剂,20mg/L的潮霉素作为转化后子叶节的筛选临界浓度,最后采用新鲜外植体,菌液中添加100μM的乙酰丁香通,浓度调整为OD值为0.5,侵染时间为10min,获得的GUS染色率较高,所有GUS染色过的植株经过PCR反应均能扩增出目的条带。研究结果为今后黄麻基因工程育种提供了重要的途径。1.2黄麻尿苷二磷酸葡萄糖焦磷酸化酶基因(UGPase)基因克隆与功能鉴定成功分离了黄麻尿苷二磷酸葡萄糖焦磷酸化酶基因(UGPase),暂命名为CcUGPasel,该基因cDNA全长1,395bp,编码465个氨基酸,编码蛋白的分质量51.15619kD,理论等电点是6.11,CcUGPase生物信息学分析表明,该基因属于糖基转移酶家族成员,RT-PCR分析表明,该基因在根、茎皮、叶中均有表达,其表达量为茎皮>根>叶,在40天和120天时期表达较高,在拟南芥中过量表达CcUGPasel基因表明,转基因植株生长速率加快、高度增加,纤维素含量比对照增加(8%-17%),木质素含量和节间距没有明显变化。表明该基因在植物的生长发育和纤维素代谢过程中起重要的调控作用。1.3黄麻纤维素合成酶基因(CcCesA1)克隆与功能鉴定克隆黄麻纤维素合成酶CcCesA1基因除5’端500bp序列外的全部cDNA。其序列长度为2529bp,编码627氨基酸残基,经Blast基因比对和蛋白质结构分析,确定是黄麻纤维素合成酶基因。半定量RT-PCR分析表明,CcCesA1在植株不同部位表达量具组织差异性,依次为茎部韧皮>根>叶>顶芽>麻骨。利用CcCesA1基因部分cDNA序列和3’UTR区,构建黄麻CcCesA1基因反义载体,用测序验证的阳性质粒转化模式植物拟南芥。Southern结果表明,外源基因以单拷贝方式整合进入基因组,转基因拟南芥生长严重受阻,植株变得矮小且茎部易弯曲倒伏,角果数量变少,长度变短,纤维素含量有不同程度的降低,本研究结果表明,所克隆的黄麻CcCesA1基因除了参与植物其他生理代谢过程外,还参与纤维素的生物合成。1.4黄麻咖啡酰-辅酶A甲基转移酶(CCoAOMT)基因克隆与功能鉴定成功分离了黄麻咖啡酰-辅酶A甲基转移酶(CCoAOMT)基因,暂命名为CcCCoAOMT1,该基因cDNA全长609bp,编码202个氨基酸,编码蛋白的分质量22.59887kD,理论等电点是5.59,CcCCoAOMT1生物信息学分析表明,该基因属于PLN02589Methyltransf-3氧甲基转移酶家族成员,RT-PCR分析表明,该基因在根、茎皮、麻骨、叶中均有表达,其表达量为麻骨>根>茎皮>叶,转基因拟南芥研究结果表明,携带CcCCoAOMT1基因的拟南芥与对照相比植株高度、角果长度、茎秆硬度增加,木质素含量比对照增加(17%-21%),表明该基因在植物的生长发育和木质素代谢过程中起关键调控作用。1.5尿苷二磷酸葡萄糖焦磷酸化酶基因(TGPase)转化黄麻研究以农杆菌介导转化无菌黄麻子叶节,提取所有转化植株叶片DNA,对转化植株进行PCR和Southern杂交检测,最后共确定有7株为转基因阳性植株,对转基因当代植株分析表明,与对照相比转基因的植株高度明显增加,韧皮纤维中纤维素含量与对照相比增加(18%-22%)。该研究结果为利用UGPase基因改良黄麻品质提供了新的视角。1.6黄麻(CCoAOMT)基因RNAi载体和人工定向沉默载体构建及转化黄麻研究利用韧皮部特异启动子启动GUS基因在烟草中的表达结果表明,与35S相比,韧皮部特异启动子可驱动GUS基因在韧皮部中表达,而在其他部位不表达或表达量较低。利用TCK303质粒和PNW55模板质粒分别构建了CCoAOMT基因RNA干扰载体和人工1niRNA定向沉默载体,农杆菌介导方法转化无菌黄麻子叶节,初步PCR检测结果表明外源基因已整合黄麻基因组中,Southern杂交结果正在检测中,有望筛选出黄麻CCoAOMT基因被定点抑制的转化植株,为后期研究重要材料。更多还原

【Abstract】 Jute (Corchorus capsularis L.), also called green hemp, belong to tiliaceae, jute genera annual herb bast fiber crops, is one of the most important long staple crops in the world, the yield and planting area are second only to cotton. Because high fiber production, luster, good moisture absorption performance, quickly aproll, easy degradation, the texture of fiber is soft, which is called gold fiber in business. In recent years as breeders of continuous efforts, jute fiber production has improved dramatically, but due to low content of cellulose and high lignin content in the jute, cause the jute bast fiber cell wall lignification, single fiber cell is shorter, the texture of fiber is hard, elastic smal, lower spinning count, seriously affect the jute fiber application in textile industry and economic benefits.Plant tissue culture is the important foundation of genetic engineering, in most plants, callus is direct receptor of Agrobacterium mediated genetic transformation, also the method to obtain protoplast, somatic hybridization and plantlet regeneration. At present there are a few research reports about jute tissue culture and regeneration system. Which seriously restricts the development of jute genetic engineering.The main research content is:optimized RNA extraction sisterm using different company extract kits and methods, established high-quality RNA extraction method of jute, using optimized5’end method, we isolated the UGPase, CesAl and CCoAOMT gene and analyzed its function. Research different medium and hormone influence on tissue culture regeneration. Using phloem specific promoter to directional inhibition CCoAOMT gene. Transfer the UGPase and CCoAOMT gene into jute, tudy the possibility to improve the fiber quality of jute using UGPase and CCoAOMT gens, the main results were as follows:1.1The establish of plant regeneration and genetic transformation system of juteTake cotyledon and caulicle of "jute179" as explants to study the callus induction. The best callus induce medium respectively is:MS+0.5mg/L TDZ+0.1mg/L or0.5mg/L IAA, MS+2.0mg/L6-BA+0.5mg/L IAA and MS+0.25mg/L TDZ +0.1mg/L or0.5mg/L NAA, MS+0.5mg/L6-BA+0.5mg/L NAA. redifferentiation culture medium of cotyledon callus is MS+0.5mg/L6-BA+0.5mg/L NAA, redifferentiation culture medium of caulicle callus is MS+0.5mg/L TDZ+2.0mg/L IAA. We obetain good result using0.1%phytic acid as the browning inhibitor. The best culture medium of bud induction was MS+2.0mg/L6-BA+0.25mg/L NAA+100mg/L HC, the rate induced is36.4%, the best medium of root induction adventitious bud is MS+0.5mg/L6-BA+1mg/L NAA. Section200mg/L Cef as for inhibited the growth of the Agrobacterium after transformation,20mg/L hygromycin as screening concentration of cotyledon. At last we use fresh explant, adjusted concentration of Agrobacterium to OD600=O.5and add100μM/L AS before30min, infection time is10min, after2days, the rate of GUS staining is higher, all GUS staining plants could amplified fragment after PCR reaction. The results provides an important way for genetic engineering breeding of jute.1.2The clone and functional identification of UDP-glucose pyrophosphorylase geneIn this study we successfully cloned the full-length cDNA of the jute UDP-glucose pyrophosphorylase gene, designated CcUGPasel, using homologous cloning and modified RACE techniques. RT-PCR analysis revealed tissue difference (stem>root>leaf) and temporal differences in CcUGPasel expression, with the highest expression levels at40d and120d. Overexpression of CcUGPasel revealed increased height and more rapid growth rate in transgenic Arabidopsis compared with control lines. Importantly, the cellulose content of transgenic Arabidopsis was increased compared with control lines, although the lignin content remained unchanged. Our results indicate that jute UGPase participates in cellulose biosynthesis. These data provide an important basis for the application of the UGP gene in the improvement of jute fiber quality.1.3The clone and functional identification of Cellulose synthase(Ces41) geneWe took stem bark of jute cultivar179(Corchorus capsularis L.) as materials, successfully cloned the full-length cDNA of jute cellulose synthetase gene except500bp of5’ terminal, using homologous cloning and modified RACE techniques. The sequence length is2529bp, encoding a627amino acids protein. Gene alignment and protein structure analysis showed that it belongs to jute cellulose Synthetase gene family. Semi-quantitative RT-PCR analysis indicated that the expression level of CcCesAl gene in different parts of plant was bark> root> leaf> bud> sticks. Using partial cDNA and3’UTR region of CcCesA1gene, constructed the antisense vector of the jute CcCesAl gene, the positive plasmids were transformed into the model plant Arabidopsis thaliana. Southern blot analysis showed that the exogenous genes were transformed into Arabidopsis genome as one copy. The growth of tansgenic Arabidopsis was badly inhibited so that plants became dwarf with easing bending stem, shorter silique, and less silique numbers. This finding shows that CcCesAl gene is involved in not only cellulose synthesis, but also other plant growth process.1.4The clone and functional identification of CCoAOMT geneThe Caffeoyl-CoA3-O-methyltransferase(CCoAOMT) is a key enzyme in lignin biosynthesis in plants. In this study we cloned the full-length cDNA of the Caffeoyl-CoA3-O-methyltransferase(CCoAOMT) gene from jute using homology clone (Primers were designed according to the sequence of CCoAOMT gene of other plants), and a modified RACE technique, subsequently named "CcCCoAOMT1" Bioinformatic analyses showed the gene is a member of the CCoAOMT gene family. Real-time PCR analysis revealed that the CcCCoAOMT1gene is constitutively expressed in all tissues, and the expression level was greatest in stem, followed by stem bark, roots and leaves. In order to understand this gene’s function, we transformed it into Arabidopsis thaliana; integration (one insertion site) was confirmed following PCR and southern hybridization. The over-expression of CcCCoAOMT1in these transgenic Arabidopsis thaliana plants resulted in increased plant height and silique length relative to non-transgenic plants. Perhaps the most important finding was that the transgenic Arabidopsis plants contained more lignin (20.44-21.26%) than did control plants (17.56%), clearly suggesting an important role of CcCCoAOMT1gene in lignin biosynthesis. These data are important for the success of efforts to reduce jute lignin content (thereby increasing fiber quality) via CcCCoAOMT1gene inhibition.1.5The research on jute transformation using UDP-glucose pyrophosphorylase geneUsing Agrobacterium mediated transformation of cotyledon of jute, extract DNA from leaves of all transformed plants, after PCR and Southern hybridization detection, select7strains positive plants, we analyzed transgenic plants results showed that compared with the control lines, the transgenic plants exhibt plant higher and more content of phloem fiber cellulose than control. The results provides a new perspective to improve fiber quality of jute with UGPase gene.1.6The RNAi and miRNA silence vector construction of CCoAOMT gene and transformationThe expression result of phloem specific promoter in the tobacco show that compared with35S, phloem specific promoter can drive GUS gene expression in the phloem, and do not express or the level expression is less. Using TCK303vector and PNW55as template plasmid constructed CCoAOMT gene RNA interference vectors and artificial miRNA directional silence vector. Transformated cotyledon of jute using verified vector, at present the result of transformation are detecting, we expected to seclect transgenic plants, provide important materials for next study.更多还原