【作者】 何玮毅；

【导师】 陈晓静； 潘东明；

【作者基本信息】 福建农林大学 ， 果树学， 2009， 博士

【摘要】 番木瓜采后贮藏期间和鲜切加工后的迅速软化所导致果实的腐败变质,已成为制约其商品化生产的重要因素。在前人对番木瓜进行采后生理研究与反义ACS和ACO基因遗传转化的基础上,本文从中锁定了与番木瓜果实后熟软化密切相关的关键细胞壁水解酶β-GAL,从分子水平再次探讨了其与番木瓜果实软化的关系。通过构建含果实特异性启动子的RNAi双T-DNA植物表达载体,经由农杆菌介导转化番木瓜胚性愈伤组织,获得了共转化转基因再生植株,为进一步选育生理上可以正常成熟、且适于鲜切加工的无选择标记基因抗软化转基因番木瓜奠定基础。主要结果如下:1.建立了适于番木瓜遗传转化的体胚发生系统。以‘漳红’番木瓜组培苗的叶片和叶柄为外植体,在改良MS + 0.5 mg·L^-1 KT + 1.0 mg·L^-1 2, 4-D + 0.5 mg·L^-1 BA + 0.1 mg·L^-1 NAA + 400 mg·L^-1 Glu + 30 g·L^-1蔗糖的固体培养基上诱导出了胚性愈伤组织。其中,CⅡa型胚性愈伤在一定条件下能够继代增殖,并能向CⅡb型转变,CⅡb型胚性愈伤则容易发生体胚。采用两步生根法,将胚性愈伤所形成的体胚再生植株先接种于1/2 MS +0.5 mg·L^-1 IBA + 1.0 g·L^-1 AC + 30 g·L^-1蔗糖的固体培养基中暗培养7天后,转移至1/2 MS + 1.0 g·L^-1 AC + 25μM·L^-1 VB12 + 30 g·L^-1蔗糖的固体培养基上光照培养,生根效果最好。移栽成活率可达45%以上。2.克隆并分析了番木瓜果肉中第一类β-Gal基因。通过一对简并引物,研究番木瓜果实后熟软化过程中β-Gal基因家族表达水平的总体变化趋势,从分子水平证实了β-GALs与番木瓜果实软化的相关性,并从50%成熟度果实中克隆了果肉加速软化时表达丰度最高的第一类β-Gal基因。分离了该类β-Gal基因4501 bp的基因组序列,经比对,其共含有17个内含子,外显子部分核苷酸序列与GenBank中的cDNA只有1个碱基的差异。生物信息学分析结果表明,该番木瓜β-GAL属于糖苷水解酶超级家族42中家族35的成员,在进化过程中与拟南芥具有较近的亲缘关系,与鳄梨和北美云杉则关系较远。同时,它还具有一段定位于胞外的信号肽,推测它可能参与细胞壁的降解。3.分离了番木瓜第一类β-Gal基因启动子,并进行了初步的功能鉴定。利用反向PCR技术,分离了第一类β-Gal基因1143 bp的5’端调控序列。在线预测结果表明,该片段含有核心启动子元件TATA盒,转录起始位点在起始密码子上游-133处。同时,还发现了与如乙烯等植物激素和胁迫应答元件,根部和胚胎器官特异性元件,以及增强子区域。利用该片段取代pCAMBIA 1301载体上GUS基因前的CaMV 35S启动子,经由农杆菌EHA 105介导侵染番木瓜不同组织器官,发现其具有明显的启动子功能,为伤诱导类型,并与特定器官的发育相关。GUS活性在果肉中最强,其次为胚和根部,其它器官中则不表达。4.构建了用于番木瓜遗传转化的植物表达载体。将β-Gal基因保守区反向重复插入载体pKANNIBAL构建RNAi中间表达载体pKAN/RG。将其上发夹结构取代经改造的载体pCAMBIA 1300上hptⅡ基因,构建中间表达载体p1300-/MFRG。分离其上单T-DNA区段,与载体pCAMBIA 2301构建RNAi双T-DNA植物表达载体p2301/TTRG。将两端含有BamHⅠ和SalⅠ粘性末端的β-Gal基因启动子片段、p2301/TTRG经XbaⅠ和BamHⅠ双酶切后的大片段和p2301/TTRG经XbaⅠ和SalⅠ双酶切后的小片段相连接,构建含有果实特异性启动子的RNAi双T-DNA植物表达载体。酶切分析和PCR检测表明,p2301/TTRG和p2301/BPTTRG均被成功导入农杆菌EHA 105,可用于后续的遗传转化研究。5.开展了以携带植物表达载体p2301/TTRG的农杆菌EHA 105介导的番木瓜遗传转化方法和体系优化的研究。探索了液体振荡转化法、浸泡转化法、体胚针刺法和茎段转化对番木瓜转化效率和共转化效率的影响,以前两种方法的效果较好,以浸泡转化法的“100μmol·L^-1 AS +菌液OD为0.2 +侵染20 min +共培养2 d”为本试验的最佳转化组合;“100μmol·L^-1 AS +菌液OD为0.2 +侵染20 min +共培养3 d”为最佳的共转化组合。后两种方法则不太适于番木瓜的遗传转化。最终,获得了5株转基因再生植株,经PCR和Southern杂交检测结果表明,只有2株为共转化的结果,诱导了其中1株生根并移栽。6.开展了番木瓜转BPTTRG基因和转MFRG的初步研究。利用浸泡转化法,以携带植物表达载体p2301/BPTTRG和p1300-/MFRG的农杆菌EHA 105,分别侵染CⅡb型胚性愈伤组织。前者获得了经GUS染色、PCR检测和Southern杂交结果为阳性的共转化转基因番木瓜再生植株。后者从198株再生植株的62个DNA混合池中,PCR检测出了两个阳性池。进一步对混合池中的单株分别进行PCR检测,则未能成功分离出转基因植株,在不含抗生素选择压的条件下的遗传转化可能出现了嵌合体现象。以上研究获得的双T-DNA共转化的转RNAi-β-Gal基因番木瓜植株,有望通过一次有性杂交,选育无选择标记基因的抗软化转基因番木瓜,在一定程度上解决转基因食品的安全性问题。更多还原

【Abstract】 The main factor limiting the commercial production of Carica papaya L. lies in the deterioration of fruit caused by rapid softening during postharvest storage and fresh-cut processing. In this paper, the key hydrolytic enzymeβ-GALs, which were responsible for the cell wall degradation and thus the fruit softening during postharvest ripening of papaya, were aimed at for the determination of relationship between its expression and fruit softening at the molecular level, on the advances of researches of postharvest physiology and genetic transformation of antisense ACS and ACO gene for papaya. The co-transformation transgenic papaya was obtained using co-cultivation of embryogenic calli with Agrobacterium tumefaciens harboring a RNAi-two-T-DNA plant expression vector driven by promoter characterized with fruit specificity, which facilitated the breeding of softening-resistant and marker-free transgenic papaya ripening normally and good for fresh-cut processing. The main results were as follows:1. Establishment of papaya embryogenesis system for genetic transformation. Explants of leaves and petioles of in vitro culture“zhanghong”papaya plantlets were inoculated on the modified MS media supplemented with 0.5 mg·L^-1 KT and 1.0 mg·L^-1 2, 4-D and 0.5 mg·L^-1 BA and 0.1 mg·L^-1 NAA and 400 mg·L^-1 Glu and 30 g·L^-1 sucrose and two kinds of the embryogenic calli were induced. Embryogenic calli of CⅡa type could be subcultured and multiplied and turn into CⅡb type, which were prone to forming somatic embryo. Two-step method for rooting showed the best rooting induction rate. Regenerated plantlets via somatic embryogenesis were firstly inoculated on the 1/2 MS media containing 0.5 mg·L^-1 IBA and 1.0 g·L^-1 AC and 30 g·L^-1 sucrose in dark for 7 days, and then transferred onto 1/2 MS media containing 1.0 g·L^-1 AC and 25μM·L^-1 VB12 and 30 g·L^-1 sucrose. After transplantation, the viability rate reached over 45%.2. Cloning and analysis of the first kind ofβ-Gal gene in papaya pulp. The total change tendency of expression level ofβ-Gal gene family during ripening and softening of papaya fruit was determined using a pair of degenerate primers, indicating the close relationship betweenβ-GALs and fruit softening. And the first kind ofβ-Gal gene cDNA with the highest expression abundance at the stage of 50% maturity, when the fruit pulp became rapid softening, was cloned. A 4501 bp genomic sequence coding for this gene was isolated, containing 17 introns and exons being one base different from the cDNA sequence. Bioinformatic analysis of this gene revealed that the protein belonged to 35 family of glycoside hydrolyase 42 superfamily, genetic relationship of which was closer with Arabidopsis thaliana and further with Persea americana and Picea sitchensis. Additionally, the predicted protein included a signal peptide located extracellularly, indicating the possible involvement of this enzyme in the degradation of cell wall matrix thus in the fruit softening.3. Isolation and preliminary characterization of the promoter of first kindβ-Gal gene. A 1143 bp of 5’ regulated sequence of the first kindβ-Gal gene was isolated using inverse PCR. Online database prediction identified core promoter elements of TATA box and a predicted transcription start at -133 upstream of the start coden, motifs for responsiveness of phytohormone especially for ethylene, and stress, and for organ specificity, and enhancer region. Characterization of this putative promoter which, by driving GUS on pCAMBIA 1301 and using Agrobacterium tumefaciens co-cultivation with different organs, was wound-inducible and organ development-related, revealed that the GUS activity was the most in fruit pulp, moderate in embryo and root and none in other organs tested.4. Construction of plant expression vectors for the genetic transformation of papaya. Conserved region ofβ-Gal gene, which coded for a key enzyme ofβ-galactosidase involved in the cell wall degradation, with the highest expression abundance at the stage of rapid softening of papaya pulp was cloned. The RNAi intermediate expression vector of pKAN/RG was constructed containingβ-Gal genes in an inverted repeat orientation with the help of pKANNIBAL vector. hptⅡgene of the modified pCAMBIA 1300 vector was replaced by the hairpin structure of pKAN/RG, which resulted in the formation of intermediate expression vector of p1300-/MFRG. Single T-DNA region of p1300-/MFRG was isolated and incorporated into the pCAMBIA 2301 vector to produce the RNAi Two-T-DNA plant expression vector of p2301/TTRG. The transformation of p2301/TTRG into Agrobactrium tumefaciens EHA 105 was confirmed by restriction enzyme analysis and PCR assay. Embryogenic calli of papaya which showed kanamycin resistance and GUS positive were obtained via genetic transformation.5. Establishment and optimization of methods for the papaya genetic transformation mediated by Agrobacterium tumefaciens EHA 105 harboring plant expression vector p2301/TTRG. Methods of shaking in liquid media, infusion, pricking, and stem-infection for papaya genetic transformation were compared and optimized according to their efficiency of transformation or co-transformation. The first two methods both applied to the regeneration of transgenic papaya, with the optimized protocol that the concentration of 100μmol·L^-1 AS for the preparation of Agrobacterium before diluting culture to OD 0.2 and the time of 20 min for infection and of 2 d for co-cultivation, for transformation; and the one consisting of 100μmol·L^-1 AS and OD 0.2 and 20 min and 3 d, for co-transformation. However, the last two methods did not work well. Five regenerated transgenic plantlets were obtained, of which two were proved to be the result of co-transformation using PCR assay and southern blot, and a co-transformed one was induced to root and transplanted.6. Regenerated of BPTTRG-transformed plantlet and a preliminary study on the transformation of MFRG in papaya. CⅡb type of embryogenic calli were co-cultivated with Agrobacterium tumefaciens EHA 105 harboring plant expression vector p2301/BPTTRG and p1300-/MFRG, respectively, using infusion method. The BPTTRG transformation produced a co-transformed transgenic papaya showing positivity in GUS staining, PCR assay and southern blot. DNAs of the 198 regenerated plantlets from the MFRG transformation were extracted and divided into sixty-two multiple pools. Of two positive pools after PCR assay, none of individual plantlet from either pool was PCR positive, possibly indicating the chimeric phenomenon occurred during the co-transformation of papaya without selection pressure of antibiotics.In summary, the two-T-DNA co-transformed transgenic papaya with a RNAi-β-Gal structure introduced into genome was obtained. For the subsequent work, it is promising to breed a softening-resistant and marker-free papaya through sexual hybridization of the co-transformed transgenic plants, for the issue of safety of transgenic food.更多还原