小麦籽粒淀粉生物合成及淀粉分支酶基因(SBE-IIb)的克隆与表达

分类号：密级：学号：2009207009 单位代码：10759石河子大学博士学位论文小麦籽粒淀粉生物合成及淀粉分支酶基因（SBE-IIb）的克隆与表达学位申请人王自布齐军仓教授指导教师李卫华教授申请学位门类级别农学博士学科、专业名称作物遗传育种研究方向荒漠绿洲区作物高产优质性状的遗传育种所在学院农学院中国〃新疆〃石河子2012年03月Starch Biosynthesis and Cloning and Expression of Starch Brangching enzymeⅡb (SBE-Ⅱb) in Wheat EndospermA Dissertation Submitted toShihezi UniversityIn Partial Fulfillment of the Requirementsfor the Degree ofDoctor of AgricultureByWang Zi-bu(Crop Breeding and Genetics)Dissertation Supervisor: Prof. Qi Jun-cangProf. Li Wei-huaMarch, 2012石河子大学学位论文独创性声明及使用授权声明学位论文独创性声明本人所呈交的学位论文是在我导师的指导下进行的研究工作及取得的研究成果。

据我所知，除文中已经注明引用的内容外，本论文不包含其他个人已经发表或撰写过的研究成果。

对本文的研究做出重要贡献的个人和集体，均已在文中作了明确的说明并表示谢意。

研究生签名：时间：年月日使用授权声明本人完全了解石河子大学有关保留、使用学位论文的规定，学校有权保留学位论文并向国家主管部门或指定机构送交论文的电子版和纸质版。

有权将学位论文在学校图书馆保存并允许被查阅。

有权自行或许可他人将学位论文编入有关数据库提供检索服务。

有权将学位论文的标题和摘要汇编出版。

保密的学位论文在解密后适用本规定。

研究生签名：时间：年月日导师签名：时间：年月日摘要小麦在我国是仅次于水稻的第二大重要的粮食作物，其产量与品质是直接关系着人类食物的供应程度和营养水平。

小麦籽粒的主要成分是淀粉，淀粉是以碳水化合物的形式储存的，人类80 %左右的能量都是由淀粉提供；淀粉除作为食品外，在工业方面作为原材料的应用更加广泛：造纸、纺织、能源、化工、制药、机械、建筑等行业对淀粉的需求潜力很大，若小麦育种能够向专业化发展，那么不同的行业对淀粉的需求必将会再上一个台阶。

因此深入研究小麦籽粒淀粉的生物合成，为以后利用生物技术来提高淀粉的产量及改良其品质奠定基础，对选育淀粉以及品质专用型的小麦具有重要意义。

为此，本研究选用不同基因型的两个高淀粉和两个低淀粉含量的六倍体小麦为材料，对高、低淀粉含量的小麦品种籽粒灌浆过程中淀粉含量积累的动态变化、淀粉合成关键酶活性的动态变化、淀粉合成关键酶基因表达差异、同时对酶活性的动态变化与淀粉积累动态以及酶基因表达差异之间存在的关系进行了相关性分析。

并且通过反转录的方法克隆得到了高淀粉小麦籽粒胚乳的SBEⅡb 的cDNA，并构建了蛋白表达载体，在大肠杆菌中诱导进行过量表达并对其表达条件进行了优化，还分别构建了SBEⅡb 基因的超量表达载体和SBEⅡb 基因的RNAi 干扰表达载体，并在模式植物中系统的分析了它们的分子特征、表达特性和功能活性等。

主要结果如下：1、小麦籽粒灌浆过程中总淀粉及淀粉组分的积累动态变化，结果表明高淀粉含量的品种和低淀粉含量的品种在籽粒淀粉积累量和积累速率上均存在明显的基因型差异。

Logistic 方程拟合淀粉积累过程发现，支、直链淀粉最终积累量取决于积累启动时间的早晚和积累速率的大小，而最大积累速率出现时间的早晚对其调节作用较小。

2、小麦籽粒灌浆过程中不同类型籽粒中的GBSS、SSS、SBE 和DBE 酶活性变化均呈现单峰曲线，峰值出现在12 -24 d不等。

4 个品种的GBSS 酶活性均与直链淀粉的积累速率呈极显著正相关，与总淀粉的积累速率达到显著或极显著正相关，但与支链淀粉的积累速率的相关性不显著。

SBE 和SSS 酶活性与总淀粉的积累速率和支链淀粉的积累速率呈显著或极显著正相关，而与直链淀粉积累速率的相关不显著。

DBE 活性与淀粉积累速率的相关性不显著，淀粉合成关键酶活性间的相关性分析表明高淀粉含量的品种DBE 活性与SSS、GBSS 和SBE 达到显著性或极显著性水平，它们之间可能存在着协同作用。

3、采用实时荧光定量RT-PCR 方法，对灌浆期籽粒淀粉合成酶相关基因的转录水平进行了检测和比较，并对其表达量与相应酶活性的相关性做了分析。

结果表明，束缚态淀粉合成酶基因（GBSS）、可溶性淀粉合成酶基因（SSS）、淀粉分支酶基因（SBE）、淀粉去分支酶（DBE）酶活性均呈单峰曲线变化,利用实时荧光定量PCR 技术检测9 个基因在不同淀粉含量的4 个供试品种花后不同时期的相对表达量,结果表明，在花后6 d这些基因开始表达，在灌浆的中期花后12 -18 d不等有表达的小高峰，两个高淀粉含量的品种在不同时期的酶活性及其酶基因的相对表达量均比两个低淀粉含量的品种相对较高；这些基因表达谱与酶活性相关分析显示，除GBSS 外其它几种淀粉合成酶基因均与相应酶活性呈显著或极显著正相关，而且GBSS酶活性到达峰值时间稍迟于DBE、SSS、SBE 等酶，说明DBE、SSS、SBE 基因可能主要通过转录水平来控制籽粒淀粉的合成，而GBSS 基因可能主要通过转录后水平来控制籽粒淀粉的合成。

I4、采用RT-PCR 技术克隆了小麦胚乳基因SBEⅡb 全长cDNA 序列，同时构建了原核生物表达载体pET28（a+）-SBEⅡb，对不同的IPTG 诱导浓度和诱导时间等条件的优化结果表明，37 ℃，8 h 能诱导SBEⅡb 融合蛋白的最大量表达，在0.5 mmol/L IPTG 浓度下可以成功表达出了SBEⅡb 蛋白。

为进一步体外研究SBEⅡb的物理化学性质及催化机理奠定基础。

5、构建SBEⅡb 的RNAi 表达载体，并对其转化烟草，检测其对直链淀粉合成的影响。

以植物表达载体PZP35S 为基础，构建了由组成型启动子35S调控的SBEⅡb 基因的RNAi 植物表达载体。

实时荧光定量PCR （qRT-PCR）检测其在转基因烟草中的相对表达量，并测定转基因植株中直链淀粉的含量和支链淀粉含量的变化。

结果表明：经qRT-PCR 证实SBEⅡb 基因的表达量较对照下降，而且支链淀粉含量变化显著，直链淀粉较对照有上升但变化没有达到显著水平。

6、构建SBEⅡb 基因的过量表达载体，将克隆得到的基因SBEⅡb 构建到了植物表达载体pPZP35S 上，通过农杆菌叶盘转化法转化烟草叶片，经卡那霉素筛选、PCR 扩增鉴定证明，SBE-Ⅱb 基因已成功转入烟草细胞中，获得4 个转基因植株。

RT-PCR 半定量和实时荧光定量RT-PCR 分析表明，转基因烟草株系的叶片中有SBEⅡb 基因的表达，与对照植株相比，转SBEⅡb 基因的植株中的转录本达到了显著性的变化，但是其淀粉分支酶活性没有达到显著性变化。

关键词：小麦；淀粉；淀粉合成酶；实时荧光定量PCR；基因表达IIAbstractWheat is the second most important food crop next to rice in china, and its output and quality are directly related to the human food supply degree and nutrition level. The major components of the wheat grain is starch, which exists in the form of carbohydrate, and about 80 % of all human energy is provided by the starch. Starch out as food outside, in the industrial sector as the raw material of the application more widely: paper, textile, energy, chemical, pharmaceutical, machinery, building and so on. So many industries to the needs of the starch is a potential, especially after the professional development of wheat breeding, different industries to the needs of the starch will again on a higher level. So further research of wheat kernels starch biosynthesis for later use biological technology to improve the production and improving their starch quality lays the foundation, and the special quality of breeding starch of wheat to have the important meaning. Therefore, this research choose different genotypes of two high starch and two low starch content of hexaploid wheat for material, and the first objective of this study was to analyse the dynamic accumulation of starch, amylose and amylopectin and to determine the patterns of the activities of key enzymes involved in starch synthesis and the ralationship to the accumulation speed of starch, amylose and amylopectin during grain filling period. The second objective was to analyze expression profiles of starch synthesis genes by qRT-PCR during endosperm development in wheat. The third objective was to clone the cDNA of SBEⅡb, and then construct the original nuclear expression vector and optimize expression condition of Prokaryotic Expression in E.coil. Constructing the interference of expression vector and overexpression vector, and then in the mode of plants to analyze and their molecular features, expression characteristic and function activity, etc.1. Correlation analysis indicated that there were obvious genotype difference between lower starch content and higher starch content starch about starch accumulating rate and Kernel filling rate. The result showed that the accumulation courses of amylopectin, amylose and total starch were well fitted to the logistic equation by relating amylopectin, amylose and total starch contents against days after pollination (DAP). The simulation parameters revealed that the higher contents of amylopectin and amylose resulted from earlier initiating accumulation time and greater accumulation rate.2. The results indicated that the activities of soluble starch synthase (SSS), granule bound starch synthase (GBSS), starch branching enzyme (SBE) and starch debranching enzyme (DBE) changed in the pattern of a single peak curve during grain filling, peak value is about between 12 d and 24 d; The accumulation rate of amylose was highly significantly correlated with the activities of GBSS, and the accumulation rate of amylopectin was significantly or sharp significantly correlated with the activities of SSS and SBE; and the accumulation rate of starch was not correlated with the activities of starch debranching enzyme (DBE), among activities of the starch synthesis enzymes correlation analysis indicated that starch debranching enzyme (DBE) of higher starch content was significantly or sharp significantly correlated with the activity of SSS、SBE and GBSS, which were interacted in starch biosynthesis.III3. The expression levels of genes were detected by real-time PCR. The results indicated that the starch enzymes activity of granule bound starch synthase ( GBSS) , soluble starch synthase gene ( SSS) , starch branching enzyme gene ( SBE), starch debranching enzyme（DBE）demonstrated a single peak curve during grain filling. To test the relative expression values of the genes in nine different grain filling stages, 6 day after pollination (DAP), 9d, 12d, 15d, 18d, 21d, 27d, 24d, 30d, by real-time quantitative RT-PCR(qRT-PCR) , and the data of starch enzymes activity from 4 wheat varieties, during grain filling middle stage, 12 -18 DAP reach reached their peaks and began to drop. The expression profile showed that starch biosynthesis enzymes genes all had high expression level in earlier stage of endosperm development. The relative expression levels of GBSS, SSS, SBE and DBE in high starch wheat varieties were higher than those in low starch wheat varieties. The correlate analysis indicated that in addition to GBSS, the relative expression level s of SBE, SSS and DBE were significantly or highly correlated with the activities of SBE, SSS and DBE at 15 DAP.The time of GBSS reached peak values were later than others. These suggested that SBE, SSS, DBE may control starch synthesis at the transcriptional level, and GBSS may control starch synthesis at the post transcriptional level.4. The cDNA of SBEⅡb cDNA from wheat endosperm was cloned by RT-PCR. The SBEⅡb cDNA were inserted pET28a (+) vector by molecular biological technologies, including restriction endonuclease digestion, electrophoresis, gel reclamation DNA ligation and so on, and then transformed to E. coil BL21 (DE3). The SBEⅡb fusion protein was expressed and the expression conditions were optimized. Through the research on optimization of expression induction time and the concentration of IPTG and so on, and results showed the expression level of SBEⅡb fusion protein reached the highest for 8 hours cultured at 37℃and 0.5 mmol/L IPTG can effectively induced the expression of SBEⅡb in prokaryotic expression system. The experimental result lay a foundation for further study on the physical and chemical properties of SBEⅡb in vitro.5. To obstain RNAi expressive vector of SBEⅡb gene, this integrated in tobacco genome and studies its effect on amylase content. RNAi vector was constructed with pPZP35S and pUCm-T, and Expression vector RNAi containing 35S promoter to regulate gene expression. qRT-PCR analysis results indicated that the SBEⅡb gene expression value is lower than CK (check plants) in the leaves of all transformed plants. Comparing with the CK about starch content, the amylopectin content of the transformed plants reached significantly changes, however, the amylose showed no significant difference.6. The plant expression vector for pPZP35S was constructed. The plasmid was transformed into tobacco by leaf dish transformation, and four transgenic plants are obtained by kanamycin selecting and PCR analysis, and the results demonstrated that SBEIIb gene was transformed into tobacco cells, and gained four transformed plants, successfully. SqRT-PCR and qRT-PCR analysis results indicated that the SBEIIb gene expressed in the leaves of all transformed plants. Comparing with the check plants, the transcriptional level of the transformed plants reached significantly changes, however, the Starch branching enzyme activity showed no difference between them.Key words:Wheat (Triticum aestivum L.); Starch; Starch Synthase; Qualitative Real-Time PCR; Gene ExpressionIV目录摘要 (I)Abstract (III)目录 (V)缩略词 (VII)引言 (IX)第一章文献综述 (1)1.1 小麦籽粒的来源、结构及其组分 (1)1.2 籽粒淀粉的组成、结构及性质 (2)1.2.1 直链淀粉 (3)1.2.2 支链淀粉 (3)1.3 籽粒淀粉的生物合成 (5)1.3.1 淀粉合成酶（SS） (6)1.3.2 淀粉分支酶（SBE） (11)1.3.3 淀粉去分支酶（DBE） (13)1.4 淀粉合成同工酶及其与淀粉的关系 (15)1.4.1 SS同工酶及其与淀粉的合成 (15)1.4.2 SBE同工酶及其与支链淀粉的合成 (19)1.4.3 DBE同工酶及其与淀粉的合成 (22)1.5 籽粒淀粉合成中关键酶基因的表达 (24)1.6 淀粉品质的遗传改良 (26)1.7 本研究的目的和意义 (27)第二章含量不同的两类小麦淀粉积累及关键酶活性变化的研究 (28)2.1 材料与方法 (29)2.1.1 材料与种植 (29)2.1.2 田间取样 (29)2.1.3 测定项目、方法 (29)2.1.4 数据统计与分析 (29)2.2 结果与分析 (29)2.2.1 籽粒淀粉含量的动态变化 (29)2.2.2 淀粉合成关键酶活性的变化 (33)2.2.3 淀粉积累速率和酶活性间的相关性分析 (36)2.3 讨论 (37)2.3.1 不同淀粉类型的小麦品种籽粒淀粉的积累特征 (37)2.3.2 淀粉合成的关键酶 (37)第三章小麦籽粒胚乳淀粉合成酶基因表达及酶活性分析 (39)3.1 材料和方法 (39)3.1.1 材料 (39)3.1.2 方法 (40)3.1.3 数据统计与分析 (40)3.2 结果与分析 (41)3.2.1 RT-PCR 结果 (41)V3.2.2 9个基因在两类不同淀粉含量的小麦品种中的表达谱 (42)3.2.3 籽粒胚乳淀粉合成酶活性的差异 (44)3.2.4 淀粉合成酶基因表达相对量与对应酶活性的相关性 (45)3.3 讨论 (45)3.3.1 实时荧光定量PCR (46)3.3.2 淀粉合成酶及其酶基因的表达 (46)3.3.3 高淀粉含量品种和低淀粉含量含量品种淀粉含量差异的机理 (47)第四章小麦胚乳基因SBEⅡb cDNA的克隆与原核表达条件优化 (48)4.1 材料和方法 (48)4.1.1 材料 (48)4.1.2 试验方法 (48)4.2 结果与分析 (50)4.2.1 SBEⅡb 基因cDNA 的扩增 (50)4.2.2 阳性克隆的菌液PCR 鉴定 (52)4.2.3 SBEⅡb cDNA 原核生物表达载体构建 (52)4.2.4 诱导表达 (52)4.3 讨论 (54)第五章小麦胚乳淀粉分支酶基因（SBEⅡb）功能初步分析 (57)5.1 材料与方法 (57)5.1.1 材料与试剂 (57)5.1.2 试验方法 (58)5.2 结果与分析 (59)5.2.1 SBEⅡb基因cDNA克隆及鉴定 (59)5.2.2 pZP35S-SBEⅡb 转化烟草 (60)5.2.3 SBEⅡb基因干扰片段及正反插入序列的酶切鉴定 (61)5.2.4 SBEⅡb基因的功能分析 (63)5.3 讨论 (67)5.3.1 淀粉合成酶基因在淀粉改良中的应用 (67)5.3.2 RNAi在淀粉品质改良中的应用 (68)第六章结论与展望 (69)6.1 结论概述 (69)6.2 主要创新点 (69)6.3 展望 (70)参考文献 (71)致谢 (84)个人简介 (85)导师评阅表 (86)VI缩略词英文缩写英文名称中文名称6-BA 6-benzyladenine 6-苄基腺嘌呤Amp Ampicillin 氨苄青霉素AGPase ADP-glucose pyrophosphorylase 腺苷二磷酸葡萄糖焦磷酸化酶ADP-G Adenoslne diphosphate glucose 腺苷二磷酸葡萄糖BSA Bovine serum albumin 牛血清白蛋白bp base pair 碱基对CB Carbenicillin 羧苄青霉素DAP Days after pollination 花后天数DBE Starch-debranching enzyme 淀粉去分支酶DEPC Diethyl Pyrocarbonate 焦炭酸二乙酯DNA Deoxyribonucleic acid 脱氧核糖核酸DMSO Dimethyl sulfoxide 二甲基亚砜dNTP deoxyribonucleoside tripospate 脱氧核苷三磷酸DTT Dithiotheitol 二硫苏糖醇EB Ethidium bromide 溴化乙锭E. coli Escericia coli 埃希氏大肠杆菌EDTA Ethylenediamine tetracetic acid 乙二胺四乙酸EDTA Ethylendiaminotetraacetic acid 乙二胺四乙酸GBSS Granule-bound starch synthase 束缚态颗粒型淀粉合成酶GL Glycerin 甘油GPM Phosphoglucomutase 磷酸葡萄糖变位酶GUS Glucuronidase β-葡糖甘酸酶G-250 Coomassie brilhant blueG-250 考马斯亮蓝Hepes N-[2-hydroxyethl]piperazine-N’[2-ethanesulfonic acid]N-2-羟乙基哌嗪N’-2-乙磺酸HK Hexokinase 己糖激酶IPTG Isopropylthio-β-D-galactoside 异丙基-β-D-硫代半乳糖Iso Isoamylase 异淀粉酶kDa kilodalton 千道尔顿Kana Kanamycin 卡那霉素LB Lyria broth LB培养基MS Murashige and Skllg medium MS培养基VIINAA Naphthalene acetic acid 萘乙酸NaAC Sodium acetate 醋酸钠ORF Open Reading Frame 开放阅读框PAGE Polymeric acrylic 聚丙烯酰胺PEP Phosphoenolpyruvate 磷酸烯醇式丙酮酸PK Pyruvate kinase 丙酮酸激酶PVP Polyvinyl lpyrrolidone 聚乙烯吡咯烷酮Pul Pullulanase 极限糊精酶qRT-PCR Quantitative real-time PCR 实时定量PCR Rif Rifampicin 利福平RT-PCR Reverse transcerption PCR 反转录PCR SBE Starch-branching enzyme 淀粉分支酶SDS Sodium dodecyl sulfate 十二烷基硫酸钠SS Starch synthase 淀粉合成酶SQ-PCR Semi-quantitative polymerase chainreaction 半定量PCRTE Tris-CL，EDTA buffer Tris-CL，EDTA 缓冲液TEMED Tetramethylethylenediamine N,N,N',N'-四甲基乙二胺X-gal 5-Bromo-4-chloro-3-indolyl-β-D-galactoside5-溴-4氯-3-吲哚-β-D-半乳糖苷引言淀粉是高等植物碳水化合物的主要储存形式。