【作者】 吕军；

【导师】 安利佳； 高晓蓉；

【作者基本信息】 大连理工大学 ， 生物化工， 2011， 博士

【摘要】 由于强吸附作用、低溶解性以及难移动性等因素的影响,磷在土壤中有效性很低,因此磷成为限制植物生长的重要因子之一。在全球范围内,70%以上的可耕地面积为酸性和碱性土壤,磷在这类土壤中的存在形式,大部分为植物所不能吸收和利用的难溶性磷酸盐。为了确保植物的产量,提高磷素的利用率,全世界每年要施用的磷肥约达3千万吨,然而,在这些施用的磷肥中,由于吸附、沉降、以及转化成有机物等形式,80%的磷肥在土壤中固定下来,难以被植物吸收利用。所以,提高植物对土壤中难溶性磷的吸收能力,对于农业生态环境的改善以及农作物的增产具有十分重要的作用。现有的研究已经表明改变有机酸在植物体内的代谢水平,增加有机酸的分泌量是增强植物利用难溶磷酸盐的一种潜在分子机制。本研究从高效解磷真菌草酸青霉菌(Penicillium oxalicum)中克隆了两个有机酸代谢途径关键酶基因,柠檬酸合酶(Cit)和苹果酸脱氢酶(MDH)基因,并对其序列进行了分析。在此基础上,将Po-mCit和Po-mMDH基因分别表达在大肠杆菌和烟草中,对转化体在磷胁迫条件下的生长状况进行了研究。具体研究内容及结果如下：1.根据已知真菌的苹果酸脱氢酶(MDH)、柠檬酸合酶(Cit)基因序列的同源性设计引物,以高效解磷真菌草酸青霉菌总RNA为模板,利用RT-PCR法,获得了草酸青霉菌线粒体苹果酸脱氢酶(Po-mMDH)和柠檬酸合酶(Po-mMDH) cDNA全序列,并由此推测相应的氨基酸序列。Po-mMDH基因cDNA (GenBank accession no. FJ550602)全长1284 bp,其中5’非编码区45 bp,3’非编码区216 bp,编码区1023 bp,编码一个由340个氨基酸构成的多肽,与土曲霉(Aspergillus terreus)和烟曲霉(Aspergillus fumigatus)mMDH具有85%以上的同源性,并具有高度保守的活性中心(VTTLDVVRASRFI),N端具有23个氨基酸的线粒体导向序列。Po-mCit基因cDNA (GenBank accession no.GQ981487)全长1716bp,包括5’端非编码区96bp,3’端非编码区205bp,开放阅读框1416bp,编码一个由471个氨基酸构成的多肽,与构巢曲霉(Aspergillus nidulans)和黑曲霉(Aspergillus niger) mCit具有85%以上的同源性,并具有高度保守的活性中心(GYGHAVLRKTDPR), N端都具有31个氨基酸的线粒体导向序列。2.分别将Po-mMDH和Po-mCit基因克隆到原核表达载体pET32a中,在大肠杆菌BL21(DE3)中分别获得可溶性融合表达产物。Po-mMDH工程菌中检测到较高的MDH活性(48.7μmol/mg protein),是对照的5倍,在以磷酸钙为唯一磷源的培养液中生长,Po-mMDH工程菌发酵液中苹果酸、乳酸、乙酸、柠檬酸和草酸的含量较对照明显增加,分别是对照的2.7、3.7、1.9、2.3和9.1倍,可溶性磷含量(134.8 mg／L)也明显高于对照(39.3 mg/L)。Po-mCit工程菌的Cit的活性为24.6μmol/mg protein,是对照的11倍。在以磷酸钙为唯一磷源的培养液中生长,Po-mCit工程菌发酵液中柠檬酸、苹果酸、乳酸、乙酸、和草酸的含量较对照明显增加,分别是对照的2.3、1.6、2.9、3.5和7.6倍,可溶性磷含量(156.6 mg/L)也明显高于对照。表明Po-mMDH或Po-mCit基因的表达能够促进有机酸的合成,增强大肠杆菌对磷的溶解能力。3.利用农杆菌转化技术获得了转Po-mMDH基因烟草,经过PCR、Southern-blot和RT-PCR验证,表明外源Po-mMDH基因已整合到烟草基因组中,并在转录水平上表达。转Po-mMDH基因烟草根系中MDH活性较野生型明显提高,低可利用磷条件下M1株系的酶活最高,达到38.6μmol/mg protein,相对于野生型增长2.2倍。低可利用磷胁迫诱导根系分泌的苹果酸含量是野生型的1.3-2.9倍。在以Al-phosphate (Al-P), Fe-phosphate (Fe-P)或Ca-phosphate (Ca-P)为唯一磷源的培养条件下,转基因烟草的生长状况明显好于野生型,干物重分别是野生型的2.5、2.3和2.3倍,总磷含量分别增长3.9、3.4和3.2倍。4.利用农杆菌转化技术获得了转Po-mCit基因烟草,并经过PCR和Southern-blot和RT-PCR验证,表明外源Po-mCit基因已整合到烟草基因组中,并在转录水平上表达。转Po-mCit基因烟草根系Cit活性较野生型明显提高,在难溶磷(Al-P)条件下,转基因株系的Cit酶活性最高,其中C2株系的Cit酶活性(28.4μmol/mg protein)是野生型(8.8μmol/mg protein)的3.2倍。根系分泌的柠檬酸含量分别增长1.6、2.7、2.5和2.3倍。在难溶磷(Al-P)培养条件下,转基因株系的地上部干重和根干重明显高于野生型,总磷含量也明显提高,较野生型分别增加1.6、2.4、1.9和1.8倍。更多还原

【Abstract】 Phosphorus deficiency is considered to be one of the major limiting factors to the improvement of field crops, due to the strong adsorption, low solubility, difficult mobility of phosphorus, as well as some other reasons. Worldwide, acidic and alkaline soils make up about 70% of the arable soil. Most of the phosphorus in these soils exists as insoluble phosphate, which is largely unavailable to plant. In order to enhance the efficiency of phosphorus usage and ensure plant productivity, nearly 30 million tons of phosphorus fertilizer are applied worldwide every year, of which, up to 80% can’t be utilized by plants because of adsorption, precipitation or conversion to organic forms. So, enhancing the ability of the plant to use soil phosphorus will play an import role in improving crop production and agro-ecological environment. There has been research showing that altering the metabolism of organic acid in plants could increase the secretion of organic acid, a potential mechanism that could enhance the utilization of insoluble phosphate by these plants.In this study, the genes of two key enzymes, citrate synthase (Cit) and malate dehydrogenase (MDH), that are involved in the oganic aicd metabolic pathway were isolated from Penicillium oxalicum, and the homology and secondary structures of putative proteins were analyzed. The two genes were also overexpressed in E.coli and tobacco, and the growth response of transformants to phosphorus deficiency was studied. The research contents and the results obtained are stated in details as follows:(1) The full-length genes encoding malate dehydrogenase (Po-mMDH) and citrate synthase (Po-mCit) of P. oxalicum were cloned by RT-PCR and RACE techniques. The full-length of Po-mMDH cDNA (GenBank accession no. FJ550602) was 1284-bp long and it contained an ORF of 1023 bp, which encodes a polypeptide of 340 aa. The nucleotide sequence had an untranslated regions of 45 bp at the 5’-end, and 216 bp at the 3’-end. The deduced transcript of Po-mMDH had 85% identity with the mMDHs of Aspergillus terreus and Aspergillus fumigatus. Po-mMDH also contained a 23-aa mitochondrial-targeting signal peptide at the N-terminus. The full-length of Po-mCit cDNA (GenBank accession no. GQ981487) was 1716-bp long, containing an ORF of 1416 bp, which encodes a polypeptide of 471 aa. The nucleotide sequence had an untranslated regions of 96 bp and 205 bp at the 5’-and 3’-ends, respectively. The deduced transcript of Po-mCit had 85% identity with the mCit of Aspergillus nidulans and Aspergillus niger. Po-mCit also had a 31-aa mitochondrial-targeting signal peptide at the N-terminus.(2) The mature genes of Po-mMDH and Po-mCit were cloned into the vector pET32a, and then transformed into E. coli strain BL21(DE3) to study their expressions in prokaryotic cell. Expressions of soluble proteins were obtained in both cases. BL21(DE3) transformed with Po-mMDH showed high MDH activity (48.7μmol/mg protein), about 5-fold higher than control. When grown in media with tricalcium phosphate as the only source of phosphorus, the transformant secreted more malate, lactate, acetate, citrate, oxalate, at about 2.7-、3.7-、1.9-, 2.3- and 9.1- folds, respectively, higher than control. Soluble phosphorus content of the transformant (134.8 mg/L) also increased significantly over control. BL21(DE3) transformed with Po-mCit showed about 11-fold increase in Cit activity. When grown in medium with tricalcium phosphate as the unique source of phosphorus, the transformant secreted about 2.3-,1.6-,2.9-,3.5- and 7.6- folds more citrate, malate, lactate, acetate and oxalate, respectively, than control. The soluble phosphorus content of the transformant was significantly increased. These results show that expressions of P. oxalicum mMDH and mCit genes in E. coli could enhance organic aicds secretion and improve the phosphate solubilizing ability of the host cells.(3) The ORF of Po-mMDH was cloned into the plasmid pBI121, and transferred into tobacco via Agrobacterium-madiated method. Positive transformants were verified by PCR, Sourthern blot and RT-PCR analysis. Three T2 progenies of homozygous plants (M1, M2 and M3) each harboring a single copy of the transgenic gene were selected for further analysis to the level of MDH expression, root malate exudate, and the ability of the plant to utilize insoluble form of phosphorus. All transgenic lines showed significantly higher level of MDH activity compared to that of wild type. Malate content in the root exudation of the transgenic lines induced in response to phosphorus deficiency was 1.3- to 2.9-fold higher than that of wild type. Among these transgenic lines, one line (M1) showed the highest level of MDH activity and malate exudate. M1 also showed a significant increase in growth over wild type, with 2.5-,2.3- and 2.3-fold increases in biomasses when grown in Al-phosphate, Fe-phosphate and Ca-phosphate containing media, respectively. M1 also had better phosphorus uptake compared to wild type, with 3.9-,3.4- and 3.2-fold increases in total phosphorus content when grown in Al-phosphate, Fe-phosphate and Ca-phosphate media, respectively.(4) The ORF of Po-mCit was cloned into the plasmid pCAMBIA1301 and transferred into tobacco via Agrobacterium-madiated method. The transformed tobacco plants were verified by PCR, Sourthern blot and RT-PCR analysis. Four T2 progenies of homozygous plants (C1, C2, C3 and C4) each harboring a single copy of the transgenic gene were selected and the level of Cit expression, the ability of the plant to utilize different forms of phosphorus and growth were investigated. The levels of Cit activity in the transgenic lines were significantly increased compared to that of wild type. Cit activity of these transgenic plants determined under different phosphorus conditions (N-P, Al-P, K-P) all produced the highest level of Cit activity when grown under insoluble phosphorus condition (Al-P). The concentration of citrate exuded by the transgenic lines was also significantly increased, with C1, C2, C3 and C4 reaching about 1.6-,2.7-,2.5-and 2.3-fold, respectively, higher than wild type. Under insoluble phosphorus condition (A1-P), significant improvements in plant growth and phosphorus utilization were observed for the transgenics lines. The transgenic tobaccos accumulated more shoot biomass and root biomass. The total phosphorus concentration of transgenic lines C1-C4 was also significantly increased, reaching about 1.6-,2.4-,1.9-and 1.8-fold higher over that of wild type.The results presented in this thesis indicated that by altering the organic acid metabolism of a plant, e.g., through over expressions of malate dehydrogenase and citrate synthase, the ability of the transgenic plant to utilize insoluble form of phosphorus could be enhanced, which may enable the plant to grow in soil where phosphorus may not be so readily available.更多还原