【作者】 苗英杰；

【导师】 何光源；

【作者基本信息】 华中科技大学 ， 微生物学， 2012， 博士

【摘要】 小麦籽粒硬度是小麦的最重要的目标性状之一，它直接决定小麦的磨粉品质和烘烤品质，是小麦品质优劣的重要指标。在控制籽粒硬度Ha位点的三个基因中，Puroindoline(Pina)和Puroindoline(Pinb)是主效基因，编码相对分子量13kDa的强碱性蛋白——PINA和PINB蛋白。这两种PIN蛋白同时存在于小麦胚乳和糊粉层细胞中，并结合在胚乳淀粉粒的表面，大量存在于成熟种子中。PIN蛋白还能够有效抑制许多植物病原菌的生长，对革兰氏阴性菌，革兰氏阳性菌以及真菌具有明显的抗性，并且PINA蛋白的抗菌作用强于PINB蛋白。细菌对抗抗生素的途径主要是通过表达特定的抗性基因分解抗生素，PIN蛋白则主要通过破坏特定脂质成分的病原菌质膜发挥抗菌能力，因此植物抗菌蛋白有望解决病原菌抗药性问题，具有很好的发展潜力。成熟的PIN蛋白具有四个α-螺旋为主的二级结构，五对二硫键把整个分子结构聚合成紧密的球型。前两个α-螺旋之间存在一个含多个疏水Trp残基和碱性Lys、Asp残基的色氨酸结构域(TRD)，很多证据证明TRD与其控制籽粒硬度和抗菌功能紧密相关。本研究构建了含多个色氨酸结构域的Pina基因人工突变体ABBC和ABBBC，以期获得抗菌性更强的植物蛋白，同时探索PIN蛋白决定籽粒硬度的分子机理；通过二级结构和三级结构的预测，探索和推测PINA两个突变蛋白的分子结构。利用PCR技术，成功地扩增了小麦Pina基因并构建了其含有不同色氨酸结构域(TRD)的人工突变体ABBC和ABBBC，并将其整合到到原核表达载体pET43.1a中，实现了在原核表达菌株Rosetta-gami(DE3)中的高效可溶性表达。采用Ni-NTA亲和层析技术，获得了高纯度和高浓度的PINA、ABBC和ABBBC重组蛋白。通过最低抑菌浓度的测定和荧光显微镜计数评估了重组蛋白的抗菌性变化。结果表明ABBC重组蛋白的抗菌性均明显高于PINA重组蛋白(P<0.05)，其可能成为有潜在的高效抗菌蛋白。而ABBBC重组蛋白的抗菌性均低于PINA重组蛋白，无明显的应用价值。这一结果表明，通过增加TRD的方式可以提高PINA蛋白抗菌性，但是当TRD数量达到3个拷贝时，抗菌性能反而下降。成功将Pina/ABBC/ABBBC构建到真核表达载体pLRPT中，通过金粉包裹，基因枪轰击，转化入四倍体小麦Ofanto中，经过诱导培养，分化培养，筛选培养，春化培养和盆栽培养，获得转基因后代。使用PCR检测，Southern Blot检测，SDS-PAGE检测以及Western Blot检测鉴定出4株转基因T0代植株，并顺利生长结实。更多还原

【Abstract】 Wheat grain hardness, one of the most important agronomic traits of wheat, directlydetermines the milling and baking quality of wheat. Ha locus, consisting of Puroindolinea (Pina) and Puroindoline b (Pinb) locating on chromosome5D short arm, controls thewheat grain hardness. Pina and Pinb are the major genes of hardness phenotype,encoding two13kDa basic proteins known as PINA and PINB. Both PINA and PINBproteins exist in the developing wheat endosperm and aleurone cells, binding to thesurface of starch granules, and of great abundance in mature seeds. Besides, Both PINAand PINB can inhibit the growth of many plant pathogens, such as gram-negativebacteria, gram-positive bacteria and fungi. PINA exhibits stronger antibacterial effectthan that of PINB.Bacterial resistance to antibiotics is mainly caused by the expression of antibioticresistance genes in bacteria. PIN proteins kill pathogens by destruction the plasmamembrane of specific lipid composition. As it is difficult for bacteria to replace the entireplasma membrane phospholipid composition, the plant antibacterial proteins are expectedto prevent the drug resistance as potential antimicrobial agents.Both PIN proteins contain a conserved cysteine backbone formed by five disulfidebonds. PIN mature proteins have a four α-helical based secondary structure. Between thefirst two α-helixes there is a tryptophan-rich domain containing several hydrophobic Trpresidues and basic Lys, Asp residues. Many evidences indicate that the tryptophan-richdomain (TRD) directly involves in grain hardness and its antibacterial function.In this study, Pina artificial mutants designated ABBC and ABBBC, with two orthree TRD, were constructed in order to obtain a more effective antibacterial protein andto explore the mechanism of PIN proteins determining grain hardness. Through thesecondary and tertiary structure prediction, the molecular structures of two PINA mutants were speculated preliminarily.Pina/ABBC/ABBBC were successfully constructed into the prokaryoticexpression vector pET43.1a and highly expressed in Rosetta-gami (DE3). RecombinantPina/ABBC/ABBBC were purified to obtain high-purity and high concentration ofrecombinant protein by means of Ni-NTA Affinity chromatography. Determination ofminimum inhibitory concentration and analyses of fluorescence microscopy were carriedout to evaluate difference in antibacterial activity of recombinant proteins. The resultsshowed that the antimicrobial activity of recombinant ABBC was significantly higherthan that of recombinant PINA, demonstrating that ABBC could be potentially effectiveanti-bacterial proteins. While the antimicrobial activity of recombinant ABBBC wassignificantly lower than that of recombinant PINA, showing no obvious application.These results indicate that this approach can improve the PINA protein antibacterialactivity by increasing the TRD, but when it contains3copies of TRD in one protein, theantibacterial activity decreased.Pina, ABBC and ABBBC were successfully constructed into the eukaryoticexpression vector pLRPT. Through the gold powder package and gene gun bombardment,the vectors were transformed into tetraploid wheat cultivar Ofanto. Several transgenicdescendants were survived. Using PCR analysis, Southern Blot test, SDS-PAGE andWestern Blot test, four transgenic T0generation plants were identified.更多还原