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PSP驱动CAPD基因的植物表达载体构建及在果树中的遗传转化研究

Studies on Construction of Plant Expression Vectors Containing CAPD Gene Driven by a Phloem Specific Promoter and Genetic Transformation in Fruit Trees

【作者】 胡桂兵

【导师】 张上隆;

【作者基本信息】 浙江大学 , 果树学, 2006, 博士

【摘要】 我国是一个水果生产大国,栽培面积和产量均雄踞世界前列,但各种检疫性、毁灭性的病害(如柑桔黄龙病、香蕉枯萎病和猕猴桃溃疡病等)时刻威胁和制约着生产的发展。培育抗病品种是解决问题的根本途径,但由于抗性资源缺乏且大部分果树存在高度杂合、倍性复杂、育种周期长等问题,杂交育种等传统的抗病育种手段难以奏效。现代生物技术的飞速发展为果树抗病育种提供了新的途径,它可以获得常规育种难以得到的新类型,从而创造出新种质。迄今为止,绝大部分果树抗病基因工程育种采用的启动子是组成型的花椰菜花叶病毒35S启动子,采用的目的基因是来源于微生物或动物、没有经过密码子优化的抗性基因,安全性、专一性和表达效率存在问题。本试验针对果树上存在的一些韧皮部传导的毁灭性病害(如柑桔黄龙病、香蕉枯萎病等),首先从笋瓜中克隆了一个韧皮部特异性启动子,并根据密码子用法分析结果,重新设计和合成了对这些毁灭性病害有杀灭作用的柞蚕抗菌肽D基因,然后构建了多种由不同启动子驱动标记基因或目的基因的植物表达载体,最后利用新构建的植物表达载体在双子叶木本果树柑桔、藤本果树猕猴桃和草本果树草莓上进行了转化研究。主要研究结果如下:(1)、根据已报道具韧皮部组织特异性的笋瓜韧皮部蛋白PP2序列设计引物,以山西本地种笋瓜叶片基因组DNA为模板,用PCR方法扩增得到了长度为966bp的PP2基因启动子片段,把片段克隆入pUCm-T载体后,经蓝白斑筛选、PCR检测和酶切鉴定后,获得了新的重组质粒pUCm-PSP,测序和序列分析表明与两个已报道的片段分别有95%和99%的同源性,推测具有相似的启动子功能。(2)、采用高频密码子分析法,分别对甜橙、温州蜜柑、葡萄柚和柠檬等4种柑桔的蛋白质编码基因序列(CDS)进行了分析,计算出了柑桔同义密码子相对使用频率(RFSC),确定出了4种柑桔的高频率密码子,发现不同种类柑桔偏爱密码子稍有差别,但不同种类柑桔的密码子偏爱性是完全一致的。用同样的方法,对柑桔现有的177个CDS进行了分析,确定出了TAA、GCT、GAT、CTT、AGG、AGA和GTT等7个高频率密码子。将柑桔的密码子使用频率与人、果蝇、酵母和大肠杆菌等不同种类模式生物比较后发现,柑桔密码子的偏爱性与不同种类生物有不同程度的差异;但将柑桔的密码子使用频率与拟南芥、番茄、水稻和尖叶蕉等不同种类的植物相比,发现柑桔密码子的偏爱性与同为双子叶植物的拟南芥、番茄完全一样,而与水稻、尖叶蕉这两种单子叶植物均有较大的差异。分析结果对动物或微生物的基因在柑桔中的表达或柑桔基因在微生物中的表达以及基因克隆时设计引物具有一定指导意义。(3)、根据177个GenBank中登录的柑桔编码蛋白密码子用法的分析结果,优化并重新设计和合成了含柑桔偏爱密码子、对柑桔黄龙病等毁灭性病害有杀灭作用的柞蚕抗菌肽D基因(命名为CAPD),克隆入pUC19克隆载体并经测序验证后,获得了含新抗病基因的重组质粒pUC19-CAPD。(4)、利用基因重组技术,分别构建了pHZ01(来源于pBI121植物表达载体和pUCm-PSP重组质粒,由PSP驱动GUS报告基因)、pHZ02(来源于pCAMBIA1301植物表达载体和pUCm-PSP重组质粒,由PSP驱动GUSA报告基因)、pHZ03(来源于pCAMBIA1302植物表达载体和pUCm-PSP重组质粒,由PSP驱动GFP报告基因)和pHZ04(来源于pCAMBIA1303植物表达载体和pUCm-PSP重组质粒,由PSP驱动GFP和GUSA融合报告基因)等4个由PSP驱动不同类型报告基因的新植物表达载体。利用细胞感受态法直接将4个原始的和4个新重组的植物表达载体分别导入根癌农杆菌LBA4404、GV3101、EHA105和发根农杆菌Ri15834等4个农杆菌中,为利用转基因技术来研究PSP的功能奠定了基础。(5)、同样利用基因重组技术,分别构建了pHZ05(来源于pBI121植物表达载体和含密码子优化的抗菌肽D基因的pUC19-CAPD克隆载体,由CaMV35S组成型启动子驱动CAPD目的基因)和pHZ06(来源于pHZ05新植物表达载体和pUCm-PSP重组质粒,由PSP驱动CAPD目的基因)2个分别由组成型和韧皮部特异性启动子驱动CAPD抗病基因的新植物表达载体。利用细胞感受态法直接将2个新重组的植物表达载体分别导入根癌农杆菌LBA4404、GV3101、EHA105和发根农杆菌Ri15834等4个农杆菌菌株中,为利用农杆菌介导的遗传转化技术培育果树抗病新种质奠定了基础。(6)、基于转化体系建立所需要的高效再生体系,我们首先针对暗柳橙的不同外植体进行了离体再生研究,以期得到较适合的外植体和培养基配方。在不同梯度浓度6-BA与IBA、NAA组合的15种培养基上,对暗柳橙无菌实生黄化苗的上胚轴不同切段及子叶进行再生分化培养,确定了暗柳橙在遗传转化中较为合适的培养基配方为MT+IBA 0.5mg/L+6-BA 2.5mg/L+蔗糖30g/L+琼脂8g/L。上胚轴切段较其它部位外植体更适于用作遗传转化的受体材料。分别以含新构建的植物表达载体pHZ05或pH06的EHA105农杆菌菌液进行了暗柳橙无菌实生黄化苗的上胚轴的转化研究,在预培养、浸染、共培养之后,采用不同的筛选分化方式,确立了使用仅添加头孢霉素抑制农杆菌生长的培养基,至外植体开始进行分化后再进行抗生素梯度浓度筛选培养的方式。这种筛选方式既有利于转化芽的生长,又可减少抗生素对转化芽的抑制作用,还可以在高浓度抗生素条件下防止非转化芽的逃逸。筛选培养4个月后,得到了一批转基因抗性芽。取其中一部分大芽叶片,提取其基因组DNA,进行多种引物的PCR检测和目的基因PCR产物的测序比较,从10株转pHZ05抗性芽中得到3株转基因植株,从60株转pHZ06抗性芽中得到了11株转基因植株。(7)、以“布鲁诺”实生无菌苗叶片为外植体,首先探讨了叶盘放置方式和暗培养时间对不定芽再生的影响,建立了高效的组培再生体系;然后用根癌农杆菌介导的叶盘法,进行了不同启动子驱动CAPD基因的转化研究,经预培养、浸染、共培养和筛选处理后,获得了若干抗性芽;最后,取其中一部分大芽叶片,提取其基因组DNA,进行多种引物的PCR检测和目的基因PCR产物的测序比较,从60株转pHZ05抗性芽中得到7株转基因植株,从40株转pHZ06抗性芽中得到了12株转基因植株。(8)、采用根癌农杆菌介导法,以草莓主栽品种“丰香”组培苗叶片为外植体,分别进行了由组成型启动子和韧皮部特异启动子驱动密码子优化抗菌肽D基因的转化,经预培养、农杆菌浸染和共培养后,先进行2周不加筛选抗生素的诱芽培养,然后经Kan筛选,获得了若干抗卡那霉素植株。提取部分抗性植株的基因组DNA,进行多种引物的PCR检测和目的基因PCR产物的测序比较,从5株转pHZ05抗性植株中得到1株转基因植株,从65株转pHZ06抗性植株中得到了14株转基因植株。本试验为研究不同启动子在果树中的表达特性和利用基因工程育种手段培育果树抗病新品种提供了扎实的基础。

【Abstract】 China is a leading country in fruit industry, ranking the top of the world both in cultivation area and in total output. The development of fruit industry, however, is facing the threat from various devastating quarantine diseases such as Citrus huanglongbing, Banana fusarium wilt and Kiwifruit bacterial canker etc. The breeding of disease-resistant cultivars, therefore, is the essential approach to those problems. The conventional method, e.g. hybrid breeding, seems not working well because of the lack of suitable germplasmic resources, the complexity in genomes and ploidy and the long period for breeding. The rapid development of the modern biotechnology has provided new ways for breeding of disease-resistant cultivars, as it can produce new genotypes and germplasms that could hardly be attained by conventional techniques. To date, most of the disease-resistance genetic engineering has adopted CaMV 35S promoter for constitutive expression and the target genes come from microorganisms or animals. But in fact these genes, if not optimized in codons, have the problems of security, specificity and efficiency. This study focused on the devastating diseases conducted via phloem, e.g Citrus huanglongbing and Banana fusarium wilt. First, a phloem-specific promoter was cloned from Cucurbita maxima and, based on the codon usage analysis, a new version of Antibacterial peptide D (APD) gene that kills the pathogens of these diseases were designed and synthesized. Then the different plant expression vectors that contained marker genes or target gene driven by various promoters were constructed. Finally the newly constructed vectors were studied by transforming into dicotylous woody fruit tree -citrus, lianoid fruit tree - kiwifruit and herbaceous fruit tree - strawberry, respectively. The results were summarized below:(1). A pair of primers was designed according to the sequence of phloem protein 2 (PP2), which shows phloem-specificity from C. maxima, and a 966bp fragment of PP2 gene in the promoter region was amplified by polymerase chain reaction (PCR) using the genomic DNA of C. maxima cv. ’Shanxi Bendizhong’ as the template. The fragment was cloned into pUCm-T vector, and a new recombined vector named pUCm-PSP was obtained after confirmed by Blue/White screening, PCR and restriction digestion. Sequence analysis indicated that this fragment had 95% and 98% homology compared with two reported promoters respectively. This result suggested that it might have the same function as other PSPs.(2). Coding DNA sequences (CDS) of four different citrus species, including sweet orange (Citrus sinensis), satsuma mandarin (C. unshiu), grapefruit (C. paradisi) and lemon (C limon) were used to analyze the relative frequency of synonymous codon (RFSC), and high-frequency codons were selected by high-frequency codon analysis. The results indicate that there was little difference in codon preference among the four citrus species. With similar method, 177 CDS were used to analyze the relative frequency of synonymous codon (RFSC), and 7 high-frequency codons including TAA, GCT, GAT, CTT, AGG, AGA and GTT were revealed by high-frequency codon analysis. When the frequency of codon usage of citrus was compared to those of Homo sapiens, Drosophila melanogaster, Saccharomyces cerevisiae and Escherichia coli, we found that the codon preference was considerably different between citrus and non-plant species. While comparing with Arabidopsis thaliana, Lycopersicon esculentum, Oryza sativa and Musa acuminata, we found that the codon preference of citrus is identical to those of the dicotyledons (A. thaliana and L, esculentum), but different from those of the monocotyledons (O. sativa and M. acuminate). The findings are helpful to researches involving introducing genes from animals or microbials or vice versa and designing primers for functional gene cloning.(3). Based on the codon usage for the amino acids of 177 citrus functional proteins deposited in GenBank, a new Antibacterial Peptide D (APD) gene with citrus preference codons was designed and chemcially synthesized. The peptide has an ability to kill the phloem conducted pathogens such as Huanglongbing, a destructive and widespread disease in citrus. The new gene encoding the same peptide, referred to as CAPD, was cloned into pUC19 to generate a recombinant plasmid pUC19-CAPD, and was verified by sequencing.(4). Four new plant expression vectors were constructed by recombinant DNA technology. pHZ01 with reporter gene GUS droved by PSP was constructed after cutting two vectors pUCm-PSP and pBI121 plant expression vector; pHZ02 whose reporter gene GUSA was droved by PSP was constructed after cutting two vectors pUCm-PSP and pCAMBIA1301 plant expression vector; pHZ03, the reporter gene GFP droved by PSP was constructed after cutting two vectors pUCm-PSP and pCAMBIA1302 plant expression vector; and pHZ04 in which the fusion reporter genes GFP and GUSA were droved by PSP was constructed after cutting two vectors pUCm-PSP and pCAMBIA1303 plant expression vector. The recombined plant expression vector was transferred into Agrobacterium tumefaciens strains of LBA4404, GV3101 and EHA105 and A.rhizogenes strain of 15834 by using cell competent method, and a good foundation had been laid for further research work in expression and function of this promoter.(5). Two new plant expression vectors, pHZ05, harboring CAPD target gene driven by the constitutive 35S promoter of CaMV (35SP), and pHZ06, harboring CAPD gene driven by PSP were constructed using recombinant DNA technology. The two new vectors, which harbor the same target gene CAPD but driven by different promoters, were introduced into competent cells of A. tumefaciens strains LBA4404, GV3101 and EHA105 and A rhizogenes strains Ri15834 respectively. The study is helpful for finding a new way to develop new germplasms resistant to some diseases in fruit trees.(6). Since the transformation system requires highly efficient regeneration system, we studied the in vitro regeneration of different explants of C. sinensis (L.) Osbeck cv ’Anliu’ orange in order to get suitable medium and explants. On the 15 media with different combinations of 6-BA, IBA and NAA, redifferentiation was observed for different parts of epicotyl and cotyledons from the sterilized etiolated seedlings of ’Anliu’ orange, and a medium prescription suitable for genetic transformation of ’Anliu’ orange were obtained, i.e, MT+IBA 0.5mg/L + 6-BA 2.5mg/L +sucrose 30g/L + agar 8g/L. It was found that the epicotyl parts were better than other explants for genetic transformation. We used the cultures of Agrobacteria EHA105 containing the newly constructed expression vector pHZ05 or pHZ06 to transform the epicotyl from sterile etiolated seedlings of ’Anliu’ orange. After pre-culture, infection and co-culture, we used a medium to which only cef were added to inhibit the growth of agro-bacteria through different selection modes for differentiation. Upon the onset of differentiation of the explants, the media containing antibiotics at different concentrations were used for selecting. This method of selecting was not only good for the growth of the transformed buds but also helpful for preventing the inhibitive effects of antibiotics on buds. In addition, it could prevent the untransformed buds from escaping. After 4 months of selecting culture, a batch of transgenic resistant buds was obtained. DNA was extracted from leaves of some of the big buds for PCR detection with several primers and the products of the target gene was sequenced and compared. 3 transgenic plants were obtained from the 10 buds transformed with pHZ05 and 11 from 60 buds transformed with pHZ06.(7). Using the leaf discs from the sterile seedlings of ’Bruno’ kiwifruit as explants, we studied the effects of the placing method and the dark culture time on the adventitious buds regeneration. A highly efficient regeneration system was established. Then using leaf discs and Agrobacteria-mediated transformation method, we conducted transformation of CAPD gene droved by different promoters. After pre-culture, infection, co-culture and selective culture, some transgenic resistant buds were obtained. DNA was extracted from the leaves of some of the big buds for PCR detection with several primers and the products of the target gene was sequenced and compared. The results showed that 7 transgenic plants were obtained from the 60 buds transformed with pHZ05 and 12 from 40 buds transformed with pHZ06.(8). Using Agrobacteria-mediated transformation method and leaf discs from tissue cultured plants of ’Toyonoka’ strawberry as explants, we transformed CAPD gene droved by 35SP and PSP respectively. After pre-culture, infection and co-culture, the explants were cultured for 2 weeks in bud inducing medium without any antibiotic, then selected by using kan antibiotic. Some transgenic resistant plantlets were obtained. DNA was extracted from leaves of some plantlets for PCR detection with several primers and the products of the target gene was sequenced and compared. The results showed that 1 transgenic plants were obtained from the 5 plantlets transformed with pHZ05 and 14 from 65 buds transformed with pHZ06.This study has laid a solid foundation for researches in the gene expressions with different promoters in fruit trees and for the breeding of new fruit cultivars resistant to diseases through genetic engineering.

  • 【网络出版投稿人】 浙江大学
  • 【网络出版年期】2007年 03期
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