【作者】 胡学博；

【导师】 郑重； 宋凤鸣；

【作者基本信息】 浙江大学 ， 植物病理学， 2004， 博士

【摘要】 我们实验室在前期研究中，以苯并噻二唑(BTH)诱导水稻抗病性，用抑制性差减杂交法分离鉴定了200多个差异表达的cDNA。基因数据库同源性搜索分析表明，其中差别表达克隆HIBI-n9和BIHI-w2与已知植物蛋白磷酸酶2C(PP2C)基因具有较高的同源性，推测这两个克隆很可能编码水稻PP2C基因的部分序列。为明确这两个可能的水稻磷酸酶基因的生物学功能以及在水稻抗病性中的作用，本研究通过5’RACE和3’RACE，结合电子克隆的方法，分离到这两个水稻磷酸酶基因的全长cDNA序列，分别命名为OsBIPP2C1和OsBIPP2C2。 采用电子克隆的方法，首先在EST数据库搜索与BIHI-w2相匹配的水稻EST，并拼接成一条长的序列。根据拼接序列设计基因特异引物，从水稻cDNA文库中用PCR扩增到1900 bp的序列。测序表明，该序列含有一个1710 bp的开放阅读框(ORF)，编码的蛋白有569 aa，分子量58.7kD，等电点为4.1。预测该蛋白的前38 aa是可剪切的信号肽。序列分析表明，OsBIPP2C1蛋白含有一个蛋白磷酸酶2C的结构域，结构域中与PP2C活性相关的与金属离子结合的碱基也大多保守。该蛋白序列与拟南芥ABI2、烟草NtPP2C1、苜蓿MP2C、拟南芥AtPP2CA等蛋白分别有14.4％、16.3％、15.0％和13.3％的相似性。因此，这是一个新的PP2C基因，命名为水稻BTH诱导的PP2C 1(Oryza sativa L.BTH induced proteinphosphatase 2C 1,OsBIPP2C1)。进一步分析发现，OsBIPP2C1以单拷贝的形式存在于水稻BAC克隆OSJNBb0019D17(登录号AC116604)中，其位点在染色体22.1cM处。在距该基因12 kb处，有一个已经报道过的肌醇-3-磷酸合成酶基因(登录号AB012107)。 用0.3 mmol／L BTH悬浮液喷雾处理水稻后6 h后OsBIPP2C1即受到快速激发，相对较高的水平维持至24 h，至72 h才逐渐降低到处理前的水平。喷雾清水的对照处理仅有微量的表达，在处理时间段没有明显波动。用BTH喷雾预处理3浙江大学博士学位论文d后接种稻瘟病菌(MagnaPorthe grisea)的水稻中，‘七刀万护Zc]基因在接种后6h受到低量诱导表达，至处理后48h开始，该基因的表达骤增。仅用稻瘟病接种处理的水稻幼苗中，OsB厅)P ZCI有一定本底表达但不受稻瘟菌接种的诱导。用1mmo比水杨酸(SA)处理水稻时，乙坛刀万护ZC]基因表达不受影响，但srnnlol/LHZO:处理后12h则受到诱导。表明在抗病信号途径中乙枯召护尸ZCI基因位于SA上游但处于HZO:信号途径的下游。口‘刀井沪ZC了同时还能被盐、重金属、冷害、干旱和ABA诱导，表明该基因在逆境信号传递中的普遍角色。 将乙七刀万沪2CI基因连接到转基因载体CHF3的CaMV一355启动子下游，转化烟草，得到13株转基因株系。PCR扩增检测口招仔孕2CI基因都整合到烟草基因组中，Northem杂交能检测到大部分株系乙七刀护尸2CI的表达，并组成型表达烟草PR了基因。转‘枯丑刀￥Ucl基因Tl代增强了烟草对TMv和烟草黑胫病护句夕toPhthoraParasitic。var. nicotiana。)的抗性。不同转基因株系在4林mol/L ABA处理下的种子萌发率和株高有较大差异。但强表达的转基因株系对高盐(200~。FL NaCI)和低渗透压(400~。比甘露醇)处理的适应性更强。表明OsB万沪2CI基因与抗病性和抗逆性相关。 用类似的方法克隆到另外一个PPZC蛋白基因口‘召万沪2口，即水稻受BTH诱导的蛋白磷酸酶2C2但下“-互口tiva L.罗H加dueed梦otein妙osphatase丝2，OsBIPPZC2)。乙坛丑厅￥UCZ有两个大小不同的转录本，乙坛丑瑟沪2口一K2(简写作K2)和OsB万沪2C2一K3(简写作K3)。分析认为，两转录本是由于较大的转录第3个外显子有一个较弱的GT一AG剪接位点，剪接后形成较小的转录本。两个选择性剪接本差异103bP，因而读码框发生移码，较小转录本提前中止翻译。K2和娜O盯大小分别为114obp和765bp，编码的蛋白分别为38oaa和255aa，分子量41.8kD和27.8kD，等电点分别是9.37和8.88。这两个蛋白在大肠杆菌中得到表达，并得到纯化的蛋白。K2和K3均含有维持PPZC酶活性的保守位点，但K3缺少第262位的AsP。K2与一个来自欧洲样(凡g“:sylvatica)的FsPPZcZ有50%核酸和67%的碱基相似性。乙坛刀万沪2C2的表达模式与乙枯刀万沪2CI有些类似但存在一定差异。首先，受BTH处理的时候，其表达比OsB仔沪2CI预处理3d再接种稻瘟菌后，乙七刀护尸2口更快，在24h即达到最高峰。其次，BTH激发的速度比乙七丑序沪ZCj稍慢，但是强 浙江大学博士学位论文度更高。第三，在与供试稻瘟菌分别表现亲和不亲和互作的水稻近等基因系HSR和HSS中，非亲和品系HSR接种稻瘟菌后12h可见OsBIPPZCZ的诱导表达，而在亲和品系HSS接种稻瘟菌对乙七刀刀邓心口没有明显的诱导。这些结果表明，OsB万)P 2C2可能与水稻抗病相关。实验证明，水稻口‘刀护尸2口基因受到ABA诱导表达;低温、千旱、创伤、重金属盐、高盐等各种逆境因子的诱导。为判别在两个转录本中那一个对水稻的抗病和逆境适应中起作用，我们进行了RT一PCR。结果表明，绝大多数的时候，只检测到心转录本。而幻转录本在伤口反应中可能有极微量表达。更多还原

【Abstract】 In the previous work of our lab, over 200 differential expressed cDNA from rice induced by benzothiadiazole (BTH) were cloned and identified through suppression subtractive hybridization. Among them, HIBI-n9 and BIHI-w2 with inserts of 414 bp and 755 bp, respectively, showed significant similarities to genes encoding known plant phosphatase 2C proteins. In order to elucidate the biological function of these two putative phosphatase 2C genes and their role in rice disease resistance, this study cloned and identified full-length cDNA of the genes and designated as OsBIPP2Cl and OsBIPP2C2, respectively.The full-length cDNA of OsBIPP2Cl was isolated through a combination of virtual cloning and RACE. Rice ESTs matching to BIHI-w2 sequence were retrieved from dbEST of GenBank database and a single long sequence was assembled. The assembled sequence is of 1900 bp long and contains an open reading frame (ORF) of 1710 bp, which predicts to encode a protein of 569 amino acids with a calculated molecular weight of 58.7 kD and the isolectric point of 4.1. A signal peptide is predicted to locate from the first amino acid to the 39th amino acid at the N-terminus of the OsBIPP2Cl protein. The predicted protein sequence contains a protein phosphatase 2C domain, approximately 255 aa in length at its C terminus. In this domain, most of the amino acids that are required for binding Mg2+/Mn2+ ions to maintain catalytic activity were identified. The OsBEPP2Cl shares weak sequence identity to other PP2Cs identified so far, showing 13-16% of identity to Arabidopsis ABI2 and AtPP2CA, tobacco NtPP2Cl and Medicago MP2C. So it is a new protein, we called it OsBEPP2Cl (Oryza_sativa L. BTHjnduced p_rotein phosphatase 2C 1, OsBIPP2Cl). The OsBIPP2Cl gene exists as single copy on the rice chromosome 3. As there is no phosphatase 2C gene identified in rice so far, we therefore concluded that OsBIPP2Cl is a novel gene encoding phosphatase 2C in rice.OsBIPP2Cl was rapidly activated as soon as 6 hr after treatment by spraying with 0.3 mM BTH solution, maintained a relatively high level within 24 hr, and thereafterdeclined gradually to the normal level at 72 hr after treatment. Inducible expression of OsBIPP2Cl was detected as early as 6 hr in BTH-treated rice seedlings after inoculation with the blast fungus, Magnaporthe grisea. This inducible expression was maintained at a relatively low level within the first 24 hr after inoculation. The significant induction was observed during the second 24 hr and then declined to normal level. In contrast, no obvious fluctuation of OsBIPP2Cl gene expression was detected in water-treated rice seedlings after inoculation. Meanwhile, OsBIPP2Cl was only slightly up-regulated by treatment with SA, but was strongly induced by 5 mM H2O2 within 12 hr after treatment. Moreover, OsBIPP2Cl was also found to be induced by salts, heavy metals and ABA, implying its multiple roles in stress acclimation. This is a common characteristic of some other stress-responsive PP2Cs such as ABI1 and ABE.Coding sequence of OsBIPP2Cl was cloned to downstream of CaMV-35S promoter in the plant binary vector CHF3. Constructed vector was transformed into tobacco by Agrobacterium-mediated leaf disc transformation. A total of 13 transgenic lines were obtained through screening of antibiotic resistant re-generating seedlings and PCR detection. Northern blotting analysis showed that OsBIPP2Cl was expressed in most of the transgenic lines. Preliminary results showed the Tl plants of the OsBIPP2Cl-overexpressing lines enhanced the resistance to tobacco mosaic virus (TMV) and tobacco blank shank (Phytophthora parasitica var. nicotianae) diseases. Moreover, the transgenic Tl plants also showed an enhanced tolerance to salt and osmotic stress. The Tl generation of the transgenic lines showed a variable sensitivity to ABA as illustrated by germination rate of the seeds and seedling growth under ABA treatments.Another PP2C gene was cloned by a similar way to that used in isolation of OsBIPP2Cl and was named as OsBIPP2C2 (Oryza更多还原