【作者】 余爱丽；

【导师】 陈如凯； 张木清；

【作者基本信息】 福建农林大学 ， 作物栽培学与耕作学， 2004， 博士

【摘要】 斑茅(Erianthus arundinaceus)是甘蔗的重要近缘属植物，生长势强、抗旱、耐瘠、宿根性好，是甘蔗育种的重要基因资源。本研究在系统分析斑茅抗逆性基础上，通过同源克隆结合RACE技术、Northern杂交、原核表达等，克隆斑茅BADH基因，并研究其在抗逆方面的分子作用。结果如下： 在干旱和盐胁迫条件下，斑茅丙二醛含量增幅较拔地拉小，质膜受损小，抗氧化酶类的清除、防御膜脂过氧化的能力强、脯氨酸积累多，斑茅的抗逆性明显强于拔地拉；利用常规比色法测定拔地拉和斑茅中的甜菜碱含量，表明斑茅甜菜碱含量高达10.73 mg／100g FW，与已测定的15植物甜菜碱含量相比，斑茅的甜菜碱含量仅次于藜科植物碱蓬和盐吸，位居第三，而拔地拉不含有或痕迹量甜菜碱。 用RT-PCR和RACE技术克隆了斑茅甜菜碱醛脱氢酶基因。斑茅BADH cDNA全长1718bp，5′端290bp左右片段GC含量高达75.9％，包含一个1515 bp的开放读码框，5′端非编码区35bp，3′端非编码区165bp，理论分子量为54.905 kDa，其推演氨基酸含有十肽VTLELGGKSP和一个与酶功能有关的Cys为醛脱氢酶高度保守的序列，以及C-端信号肽SKL。将斑茅BADH编码区1515 bp核苷酸序列在GenBank中进行比较，发现其与Zea mays PCO110216的已知部分序列同源性为97％；与S.bicolor BADH1部分序列同源性为95％；与O.sativa BADH、H.vulgare BBD2和T.aestivum BADH的5′端无同源性。 分子进化分析表明，22个BADH基因由同一基因进化成五个类群，双子叶植物进化变异较大；所有已知禾本科植物斑茅、高粱、玉米、大麦、小麦的BADH基因为一簇，其中按同工酶类型分为两组；斑茅与高粱的同源性高于斑茅与玉米，以及高粱与玉米的同源性。 包含醛保守序列的斑茅BADH部分cDNA序列作探针，进行Southern和Northern杂交分析。结果表明，斑茅BADH在基因组中有一到两个拷贝；斑茅BADH为诱导型表达基因，干旱胁迫，表达量增加，复水后其表达量减少；盐胁迫时，表达量随着胁迫时间增加，然后保持某一水平，但又受环境温度影响；盐胁迫10天时，根中甜菜碱醛脱氢酶的表达量大于茎中的，茎中的大于叶中。 构建斑茅甜菜碱醛脱氢酶的原核表达载体，原核表达斑茅甜菜碱醛脱氢福建农林大学博士学位论文酶基因，在酶粗提液中检测到酶的比活力为9.04Inn。功吐n一，m扩蛋白。 斑茅BADH基因的成功分离并转导甘蔗将有利于实现甘蔗抗早基因工程，提高抗早育种效率。更多还原

【Abstract】 Erianthus arundinaceus, the related genara of Saccharum, has good applicability, stress resistance, vigour and ratooning ability. So, it is important gene resource for sugarcane breeding. Based on the comparative analysis for stress resistance of E. arundinaceus and Saccharum, BADH gene from E. arundinaceus was cloned and its expression was analysed in E.coli or under high salinity and drought stress. The results were as follows:The increments of MDA content and the plasma membrane permeability in leaves of E.arundinaceus were less than those in Badila (S. qfficinarum L.) subjected to water deficit or salt stress, and higher activities of SOD and POD in leaves of E.arundinaceus. 10.73 mg/100g FW of glycinbetaine in water-stressed E.arundinaceus was less than that of S. glauca or S. ussuriensis, but higher than those of the other reported plants. No glycine betaine accumulated in water-deficit Badila.The full-length BADHcDNA from E.arundinaceus was isolated by RT-PCR and rapid amplified cDNA ends (RACE). E.arundinaceus BADH was 1718 bp in length, including 35 bp 5’UTR, 165 bp 3’UTR and 1515-base-pair open reading frame (ORF) encoding protein of 54.905 kDa. 75.9% of G and C was found in the 290bp length of its 5’end. The deduced amino acid sequence of E.arundinaceus BADH included a decapeptide sequence "VTLELGGKSP" and a Cysteine residue, which is highly conserved among general aldehyde dehydrogenases (ALDH). It also contained a tripeptide SKL at the C-terminnal, a signal known to target to microbodies. The BLAST analysis in GenBank showed that overall coding region of E. arundinaceus BADH is c. 97% homologous to the Zea mays cDNA sequence ( PCO110216), c. 95% to the partial cDNA of Sorghum bicolor BADH1. However, its 5’end had no homology to O. sativa BADH, H. vulgare BBD2 and T. aestivum BADH.The phylogenetic analysis from 22 kinds of plant BADHs indicated the larger variability in the dicotyledonous plants, one group in the Gramineae plants including BADH genes form E.arundinaceus, S.vulgare, Zea mays, H. vulgare andT. aestivum, which branches out into two groups corresponding to betaine aldehyde dehydrogenase isozymes..Southern and Northern blot analysis were carried out using cDNA fragment of the conserved domain sequence of E.arundinaceus BADH as a probe. Southern blot showed one or two copies of E.arundinaceus BADH in the genome of E. arundinaceus. Northern blot showed the E. arundinaceus BADH was expressed at high levels under drought stress, then decreased after recovering water. However, its expression levels increased with the elongation of salt stress duration, then kept at a certain level. On the tenth day of salt stress, the level of mRNA for Karundinaceus BADH in stem of E. arundinaceus was higher than that in leaves, lower in roots.The expression vector of E.arundinaceus BADH was constructed. E.arundinaceus BADH could be expressed in E.coli BL21.The specific activity of E.arundinaceus BADH induced by IPTG was 9.04 nmolmin-1mg-1protein.Cloning of E. arundinaceus BADH gene and its successful transformation into sugarcane cultivars will be an alternative to improve drought tolerance in sugarcane in future.更多还原