【作者】 李晓薇；

【导师】 王庆钰；

【作者基本信息】 吉林大学 ， 植物学， 2011， 博士

【摘要】 大豆不仅可提供优质丰富的蛋白质和油脂,更是具有多种生物活性的异黄酮类化合物的重要来源。异黄酮被认为在植物-微生物互作系统中发挥着多种多样的作用,在人类的营养及健康方面也具有重大的潜力。大豆异黄酮是一类次级代谢产物,它们是由苯丙烷类代谢途径的一个分支所合成的,这个途径在整个植物界普遍存在。研究发现,大多数MYB转录因子对植物类黄酮的代谢均有调控作用。MYB是植物中数量最多,功能最多样化的一类转录因子,参与对植物次生代谢的调控、激素和环境因子的应答,并对细胞分化、细胞周期以及植物叶片等器官的形态建成具有重要的调节作用。因此,对大豆MYB转录因子进行研究,明确其在大豆类黄酮代谢中的地位和作用,阐明其功能,将有助于揭示大豆异黄酮的调控机理。本研究利用PCR技术首次从大豆品种吉林32中克隆了一个新的MYB基因(基因银行注册号码为JF510467),命名为GmMYB12B2,同时克隆了GmMYB12a基因,并对它们的功能做了初步分析。主要研究结果如下：1.利用RT-PCR技术从大豆品种(吉林32)中首次克隆了GmMYB12B2基因,全长783bp,编码260个氨基酸；通过氨基酸序列比对分析发现,它含有两个MYB结构域,属于典型的R2R3-MYB转录因子。同时克隆了GmMYB12a基因。2.构建GmMYB12a与GmMYB12B2基因的原核表达载体pET-28a-GmMYB12a及pET-28a-GmMYB12B2,分别转入大肠杆菌Rosetta(DE3)中,在28℃、1.2 mMIPTG诱导6h后,可以获得纯度较高的目的蛋白。3.利用半定量RT-PCR分析了GmMYB12a与GmMYB12B2基因对各种胁迫的应答情况。结果显示,在紫外辐射、高盐胁迫下,随着处理时间的延长,两基因的表达量逐渐增加,而GmMYB12B2的表达量升高的较GmMYB12a显著。两基因对低温、干旱、ABA响应不明显。4.构建酵母效应质粒pGBK7-GmMYB12a及pGBK7-GmMYB12B2,转入酵母菌株AH109中,在营养缺陷型培养基SD/-Trp/-Ade/-His+3-AT上筛选阳性转化子。酵母表达结果显示,GmMYB12B2具有明显的转录激活活性,β-半乳糖苷酶显色反应呈现出明显的蓝色；转入GmMYB12a的酵母菌不能在营养缺陷型培养基上生长,表明其不具转录激活功能或具有极微弱的转录激活活性。5.构建绿色荧光蛋白融合表达载体pBI121-GFP-GmMYB12a及pBI121-GFP-GmMYB12B2,农杆菌介导法转化洋葱表皮细胞进行瞬时表达,结果表明,两基因均定位于细胞核中,与亚细胞定位预测一致。6.利用定量PCR技术对GmMYB12a与GmMYB12B2基因的组织特异性表达情况进行了检测,结果表明,GmMYB12B2在根及成熟的种子中表达量较高,随着种子逐渐发育成熟,其表达量也随之升高；GmMYB12a与GmMYB12B2的表达模式基本一致,只是其表达量相对较低。7.利用HPLC法测定了大豆不同组织中异黄酮的含量,结果表明,大豆各组织的异黄酮含量情况为：大豆籽粒>60d胚>50d胚>40d胚>花>荚>20d胚>30d胚>叶>根>茎。未成熟胚中大豆异黄酮的积累基本符合随着成长天数的增加而增加的规律,只是在30天胚中有所下降,这一积累规律与GmMYB12a和GmMYB12B2基因在这些时期的表达规律基本一致。8.构建植物表达载体pCAMBIA1301-CHS8P、PPZP-GmMYB 12a及PPZP-GmMYB12B2,按照不同组合在大豆愈伤组织中进行瞬时表达。CHS8是植物类黄酮合成途径中的关键酶,通过分析MYB转录因子是否与其相互作用可以鉴定MYB是否参与了类黄酮的生物合成。实验结果表明,共侵染pCAMBIA1301-CHS8P与PPZP-GmMYB 12B2质粒载体的大豆愈伤组织的GUS荧光值最高。这就表明,转入GmMYB12B2基因确实对CHS8基因有调控作用,使共转化的大豆愈伤组织GUS荧光活性升高。GmMYB12a的作用不明显。9.构建植物表达载体PPZP-GmMYB 12a及PPZP-GmMYB 12B2,利用农杆菌介导法转化拟南芥。对转基因拟南芥T3代植株中类黄酮合成途径中各关键酶的表达情况进行检测,结果表明：在转GmMYB12B2的株系中,PALI、CHS、FLS的表达明显较野生型及转入空载体的拟南芥株系的表达量要高,而CHL、F3H、F3’H的表达在各株系中变化不明显,DFR的表达量在转GmMYB12B2的株系中稍有下降。GmMYB12a与GmMYB12B2的表达模式基本一致,只是其表达量相对较低。这说明GmMYB12B2与GmMYB12a均可调控植物的类黄酮的生物合成。对转基因拟南芥的抗性分析显示,两基因均可提高转基因拟南芥的盐及紫外辐射耐受能力,只是GmMYB12a的作用较GmMYB12B2弱。更多还原

【Abstract】 Soybean can provide abundant protein and oil, it also act as the main source of varies isofalvone compounds. Isoflavones are considered to play diverse roles in plant-microbe interaction and also have great potential to human nutrition and health. Soybean isoflavones are a class of secondary metabolites of the phenylpropanoid pathway which exists throughout the whole plant system. Many researches have approved that lots of MYB transcription factors could regulate the biosynthesis of isofalvones in plants. MYB transcription factors are the largest transcription factor family in plant, their functions in secondary metabolism, environmental stress, cell differentiation, cell cycle and morphogenesis of organs are all well known now. To further confirm the function of MYB transcription factors in the regulation of isofalvone biosynthesis, we cloned and characterized two MYB transcription factors in soybean.In this study, we cloned a novel MYB transcription factor from soybean (Jilin 32) and named GmMYB12B2 (GenBank accession number:JF510467). We also cloned the GmMYB12a. The function of them in regulation of isoflavone biosynthesis was discussed. The main results are as follow:1. We cloned the GmMYB12B2 transcription factor using Reverse Transcript-PCR method from soybean (Jilin32). It contains 783 nucleotides and encodes 260 amino acids, it contains two MYB domains according to sequence alignment and belongs to typical R2R3-MYB transcription factor. We also cloned the GmMYB12a.2. We constructed the pET28-GmMYB12a and pET28-GmMYB12B2 plasmids and transformed them into E. coli Rosetta (DE3). The proteins were induced in 28℃for 6 hours by 1.2 mMIPTG..3. We analysised the expression levels of GmMYB12a and Gm MYB12B2 under salt, low temperature, drought, ABA and UV radiation treatments using semi-quantitative RT-PCR. The expression of GmMYB12a and Gm MYB12B2 are all induced by UV radiation and salt treatment dramatically, especially GmMYB12B2. They are not induced by low temperature, drought and ABA treatment. 4. We constructed yeast effect plasmids pGBK7-GmMYB12a and pGBK7-GmMYB12B2, and then they were transformed into the yeast strain AH 109. The positive transformants were selected using nutrient-deficient medium SD/-Trp/-Ade /-His with 3-AT. The result showed that GmMYB12B2 have transcriptional activation, GmMYB12a have no or weak transcriptional activation.5. We constructed pBI121-GFP-GmMYB12a and pBI121-GFP-GmMYB12B2 plasmids, and then they were transformed into onion epidermal cells using Agrobacterium-mediated method. The subcellular location showed that they are all located in the nucleus.6. Real-time RT-PCR was carrying out to analyze the tissue-specific expression of GmMYB12B2 and GmMYB12a in soybean. The GmMYB12B2 transcripts were higher in root and mature seed than other organs. GmMYB12a have the same expression pattern as GmMYB12B2, but its expression levels were lower than GmMYB12B2.7. The contents of isoflavones were detected in different tissues using HPLC method. The results showed that the isoflavones contents in following organs were:soybean seed> 60d embryo> 50d embryo> 40d embryo> flower> pod> 20d embryo> 30d embryo> leaf> root> stem. The accumulation of isoflavones in immature embryos increased during plant growth. This result is consistent with the genes expression levels.8. pCAMBIA1301-CHS8P. PPZP-GmMYB12a and PPZP-GmMYB12B2 plasmids were constructed and transformed into soybean callus cells. CHS8 is the key enzyme in plant flavonoid biosynthesis, the cotransformation of MYBs and CHS8 will confirm the interaction of them. The results showed that the cells that cotransformed of pCAMBIA1301-CHS8P and PPZP-GmMYB12B2 have the highest GUS flurescence. It proved that GmMYB12B2 could regulate CHS8 to produces more fluorescence.9. We constructed PPZP-GmMYB12a and PPZP-GmMYB12B2 plasmids, and then they were transformed into Arabidopsis thaliana. The expression levels of some key enzymes in flavonid biosynthesis were dectected in T3 progeny. The results showed that:the expression levels of PALI, CHS and FLS were significantly higher than wild-type and vector in GmMYB12B2 transgenic lines, while the expression levels of CHI. F3H and F3’H is same in all lines. The expression level of DFR is lower than WT and vector in GmMYB12B2 transgenic lines. GmMYB12a have the same expression pattern as GmMYB12B2 in GmMYB12a transgenic lines, but its expression levels were lower than GmMYB12B2. These results proved that GmMYB12B2 and GmMYB12a are all regulate the flavonid biosynthesis. The transgenic lines of them have more tolerance during salt and UV radiation treatment.更多还原