【作者】 王延秀；

【导师】 张金文；

【作者基本信息】 甘肃农业大学 ， 作物遗传育种， 2010， 博士

【摘要】 紫花苜蓿(Medicago sativa L.)草质优良、营养丰富、适口性强,被誉为“饲草之王”,是亚洲、北美洲利用最广泛、最重要的豆科牧草。然而,反刍牲畜采食新鲜苜蓿后易引起臌胀病(bloat),影响到它在放牧方面的利用。一般认为,苜蓿叶片中浓缩单宁(condensed tannins, CT)含量低是引起反刍牲畜臌胀病的主要原因。通过遗传调控,促使叶片中CT合成和积累是苜蓿遗传育种的重要研究内容,具有重要的理论和实践意义。CT的生物合成是通过莽草酸途径产生的苯丙氨酸经一系列反应最终形成,由众多酶参与。二氢黄酮还原酶(dihydroflavonol reductase, DFR)是其生物合成途径中的关键酶,调控DFR基因的表达水平可以改变CT的积累,但转基因紫花苜蓿中dfr的表达水平有待进一步提高。PNZIP (pharbitis nilleu zipper)启动子具有使基因在光合组织中高效表达,而在非光合组织中表达水平很低的组织特异性启动子。为了提高紫花苜蓿叶片以及茎等家畜采食器官中CT含量、获得抗臌胀病紫花苜蓿的种质新材料,本实验开展了DFR基因和PNZIP组织特异启动子的克隆、PNZIP启动子驱动的DFR基因植物表达载体构建及对紫花苜蓿遗传转化研究,取得了如下结果：1.以蒺藜苜蓿(Medicargo trunctula L.)幼果为试材,采用RT-PCR方法克隆到二氢黄酮还原酶(DFR)基因cDNA片段。该片段全长1018 bp,编码337个氨基酸,与GenBank中已注册的DFR基因同源性为99.8%。构建DFR基因原核表达载体pETDFR, DFR在原核表达系统中得到了高效表达,获得预期大小(39.8 kDa)的酶蛋白分子,说明克隆的DFR基因编码序列完整。2.采用PCR技术,从裂叶牵牛(pharbitis nil choisy Morninga Glory)基因组DNA中克隆到1487 bp的PNZIP启动子。序列分析表明：该片段除含有一个典型的真核生物核心启动子区域(-60--10 bp)和多个TATA-box、CAAT-box等启动子元件外,还存在I-box、ACE、G-box、CAANNNNATC元件、Box-Ⅱ、CCAAT-盒等6类光效应元件,预示着在光合组织的生物代谢中具有调控作用。以绿色荧光蛋白基因(GFP)作为报道基因,构建PNZIP启动子驱动的GFP基因植物表达载体,在转GFP基因烟草的绿色组织,尤其是叶肉、茎中都得到高效表达,说明表明所克隆的PNZIP启动子具有光合组织表达的特异性。3.以pBI121为基础载体,分别构建了组成型CaMV 35S启动子、光合组织特异型PNZIP启动子驱动的DFR基因植物表达载体pBIDFR和pPNDFR。用含有pBIDFR、pPNDFR的农杆菌(EHA105)工程菌转化烟草(Nictiana tabacum L.)品种红花大金子(2n=4X=48),分别获得了27株和23株Kan抗性植株。采用正丁醇盐酸法测定了野生烟草(对照)、不同启动子驱动的DFR基因转化再生烟草的叶片、茎和根等器官中的CT含量,结果表明以组成型启动子驱动的DFR基因(pBIDFR)转化烟草各器官中CT含量显著高于对照,叶片中的含量达到4.27mg/g,比对照叶片中的CT含量高75%；而以光合组织特异表达启动子驱动的DFR基因(pPNDFR)转基因烟草的茎叶中CT含量显著高于野生型烟草,其中叶片中浓缩单宁含量达到4.01 mg/g,比对照叶片的含量高64.34%。说明PNZIP启动子的活性接近组成型启动子CaMV35S,但从能量角度考虑,PNZIP启动子驱动基因的表达模式更为经济。4.以苜蓿优良品种“中苜一号”为材料,研究了农杆菌工程菌pPNDFR/EAH105的转化体系,得出以5-7d龄子叶预培养3 d、采用OD6000.3的工程菌液侵染30 min和共培养3d的转化体系,抗性愈伤组织诱导率高达70%。获取的抗性愈伤组织经过诱导,最终获得了6株抗性转基因植株和一批抗性不定芽。更多还原

【Abstract】 Alfalfa (Medicago sativa L.) has been taken as the "king of forage" for its’ excellent forge quality, rich nutrition and strong palatability. It is the most widely used and most important legumes in Asia and North America. However, the ruminant livestock feeding with fresh alfalfa would easily cause bloat disease, this has affected its use in grazing area. It is generally accepted that low content of condensed tannins (CT) in alfalfa leaf is the major reason that caused ruminant livestock’s bloat. Through genetic manipulation, promoting synthesis and accumulation of CT in alfalfa leaves is the most important research topic of alfalfa breeding, and has great significance in theory and practice.The condensed tannin biosynthesis is through the shikimate pathway and produced phenylalanine, after the latter form the CT after series of reactions and there has many enzymes involved in it. Dihydroflavonol reductase (DFR) is the key enzyme of the biosynthesis of CT, regulating the expression level of DFR gene can change the accumulation of CT, but the expression level of dfr in genetically modified alfalfa would be further improved. PNZIP (pharbitis nilleu zipper) promoter is a tissue-specific promoter that will drive the target gene expressed in photosynthetic tissues efficiently, while in non-photosynthetic tissues at very low levels of. In order to improve the content of CT in alfalfa leaves, stems and other organs of livestock feed, achieve new germplasm materials for breeding anti-bloat alfalfa, the experiment carried out DFR gene and PNZIP tissue-specific promoter cloning, PNZIP promoter drived DFR gene plant expression vector construction and genetic transformation on alfalfa, achieved the following result:1. RT-PCR was employed to clone cDNA fragment of DFR gene from Medicargo truncatula young fruit. This fragment is 1018 bp, encode 337 amino-acid residues. Sequence analysis showed that 99.8% of the DFR were homologous to those in GenBank. The two base mutation did not affect the translation of amino-acid, based on inferred online there is only one amino acid change. DFR gene Prokaryotic expression vector named pETDFR was constructed; and DFR gene has expressed at high level in the prokaryotic expression vector, achieved the expected size 39.8 KD enzyme protein. All these indicated that the coned DFR gene has complete coding sequence.2. By PCR method, the 1487 bp PNZIP promoter was cloned from pharbitis nil choisy Morning Glory genomic DNA. Sequence analysis showed that:In addition to a typical eukaryotic core promoter region (-60--10 bp) and a number of TATA-box, CAAT-box and other promoter elements, there are 6 light effect elements such as I-box, ACE, G-box, CAANNNNATC components, Box-II, CCAAT-box in it, indicating the sequence has a regulatory role in photosynthetic tissues microbial metabolism. Taking green fluorescent protein (GFP) as reporter gene, the plant expression vector named pPNGFP which GFP gene was drived by PNZIP promoter was constructed, in transgenic tobacco green tissue especially in mesophyll and stem GFP gene have been expressed efficiently, indicating that the cloned PNZIP promoter has the specificity expressed in photosynthetic tissue3. Based on pBI121 vector, the constitutive promoter CaMV 35S and photosynthetic tissue-specific promoter PNZIP drived DFR gene plant expression vector pBIDFR and pPNDFR were constructed. With agrobacterium (EHA105) engineered bacteria containing pBIDFR, pPNDFR transformed tobacco (Nictiana tabacum L.) varieties "Honghuadajingzi’(2n=4 X=48),27 and 23 Kan resistant plants were received respectively. Hydrochloric acid method was used to determine the CT content in wild tobacco (control), different promoter drived DFR gene transformed tobacco leaves, stems and roots organs, the results show that the constitutive promoter drived DFR gene (pBIDFR) transformed tobacco every organs’ CT content was significantly higher than the control, in leaves up to 4.27 mg/g,75% higher than the control leaves’. The photosynthetic tissue specific promoter drived DFR gene (pPNDFR) transgenic tobacco stems and leaves’condensed tannins were significantly higher than wild type tobacco, which leaves’condensed tannin contents was 4.01 mg/g,64.34% higher than the control leaves. This indicated that the PNZIP promoter activity is near to the constitutive promoter CaMV35S, from the point of energy PNZIP promoter drived gene expression pattern is more economical.Taking alfalfa varieties "Zhongmuyihao" as material, the Agrobacterium engineering bacteria pPNDFR/EAH105 transformation system was studied. Obtained with 5-7d old cotyledons as explants, pre-cultured 3 d, OD 600 0.3 engineering bacteria concentration, infected for 30 min, co-cultured 3 d transformation system, which can get 70% resistant callus induction rate. After induction the resistant callus eventually acquired 6 resistant transgenic plants and a number of resistant buds.更多还原