【作者】 金顺玉；

【导师】 卢孟柱；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2009， 博士

【摘要】 本论文采用RT-PCR结合RACE方法,从一年生实生苗毛竹中克隆了木质素合成过程中非常重要的四个相关酶基因—CCoAOMT1、CCoAOMT2、C4H、4CL的全长。运用生物信息学方法对其核苷酸序列、编码的氨基酸序列进行分析。并利用荧光定量PCR(QRT-PCR)技术对毛竹一年生根、叶、杆箨、茎、二年生茎、三年生茎、冬笋、春笋、笋顶部、笋中部、笋基部植物材料分析了这四个基因在不同发育时期、不同表达部位中表达丰度的变化,并验证了其在维管组织中特异表达特性。本论文得到以下结论:(1)毛竹CCoAOMT1基因cDNA序列全长1045bp,从第86bp有一个起始密码子到第871bp处终止密码子结束,含有一个完整的开放读码框,共编码了262个氨基酸。将该基因的蛋白序列通过在SMART网站进行分析,发现该基因属于细胞色素p450酶家族中一员。通过ExPaSy网站分析发现该基因具有15个第Ⅷ因子结构域位点标记;2个整合素beta链半胱氨酸富集区位点标记;9个表皮样生长因子位点标记; 1个4Fe-4S铁氧化还原蛋白铁硫结合区位点标记;2个2Fe-2S铁氧化还原蛋白铁硫结合区位点标记;1个过敏毒素位点标记;2个硫解酶激活位点;4个羧基端胱氨酸结位点标记;1个类生长因子N结合蛋白末端结构域信号区。与NCBI核酸数据库中序列进行比对,与绿竹相似性最高,得分均为99%,其次是禾本科的玉米和水稻,得分分别为88%、88%,与双子叶及木本植物的亲缘较远。通过荧光定量分析,该基因在毛竹不同发育时期和不同组织中表达丰度不同,在春笋顶部表达量最高,其次是春笋中部、春笋基部、杆箨、冬笋、二年生茎、一年生茎、一年生根、一年生叶、春笋和三年生茎。(2)毛竹CCoAOMT2基因cDNA序列全长1014bp,从第48bp含有一个开放读码框,第839bp处有终止密码子,共编码了264个氨基酸。将该基因的蛋白序列通过在SMART网站进行分析,发现该基因属于细胞色素p450酶家族中一员。通过ExPaSy网站分析发现该基因具有有1个整合素beta链半胱氨酸富集区位点标记;9个表皮样生长因子位点标记;15个第Ⅷ因子结构域位点标记;4个2Fe-2S铁氧化还原蛋白铁硫结合区位点标记; 2个4Fe-4S铁氧化还原蛋白铁硫结合区位点标记;3个过敏毒素位点标记; 2个硫解酶激活位点;3个羧基端胱氨酸结位点标记。与NCBI核酸数据库中序列进行比对,与禾本科单子叶的水稻、玉米相似性最高,得分分别为88%、87%,与双子叶及木本植物的亲缘较远。通过荧光定量分析,该基因在毛竹不同发育时期和不同组织中表达丰度不同,在春笋顶部中表达量最高,其次是春笋中部、冬笋、春笋基部、二年生茎、三年生茎、春笋、一年生根、杆箨、一年生茎、一年生叶。(3)毛竹C4H基因cDNA序列全长1746bp,从第72bp有一个起始密码子,到第1577bp处终止密码子结束,含有一个完整的开放读码框,共编码了502个氨基酸。将该基因的蛋白序列通过在SMART网站进行分析,发现该基因属于细胞色素p450酶,CYP73亚家族中一员。通过ExPASy网站分析,该基因序列上含有:12个表皮样生长因子位点标记;3个过敏毒素位点标记;2个羧基端胱氨酸结位点标记;6个2Fe-2S铁氧化还原蛋白铁硫结合区位点标记;12个第Ⅷ因子结构域位点标记;1个4Fe-4S铁氧化还原蛋白铁硫结合区位点标记;1个硫解酶激活位;1个整合素beta链半胱氨酸富集区位点标记;1个哺乳动物防御素位点标记。与NCBI核酸和数据库中序列进行比对,得出毛竹与禾本科单子叶的高粱、水稻和玉米相似性最高,分数分别为:88%、88%、87%,与双子叶及木本植物的亲缘较远。通过荧光定量分析,该基因在毛竹不同发育时期和不同组织中表达丰度不同,在冬笋中表达量最高,其次是春笋顶部、春笋中部、一年生叶、三年生茎、杆箨、一年生根、二年生茎、春笋、一年生茎、春笋基部。(4)毛竹4CL基因cDNA序列全长1792bp,从第90bp有一个起始密码子,到第1715bp处终止密码子结束,含有一个完整开放读码框,共编码了543个氨基酸。将4CL基因的蛋白序列通过SMART网站进行分析,发现该基因属于AMP-Binding家族中一员。通过ExPASy网站分析,该基因序列包括10个表皮样生长因子位点标记;2个过敏毒素位点标记;2个羧基端胱氨酸结位点标记;6个2Fe-2S铁氧化还原蛋白铁硫结合区位点标记;20个第Ⅷ因子结构域位点标记;2个4Fe-4S铁氧化还原蛋白铁硫结合区位点标记;4个整合素beta链半胱氨酸富集区位点标记;3个哺乳动物防御素位点标记;2个胰岛素样生长因子结合蛋白;1个Janus-faced atracotoxin (J-ACTX) family signature;与NCBI核酸数据库中序列进行比对,得出毛竹与慈竹的相似性最高,分数为100%,其次是单子叶水稻和玉米,得分分别为97%和83%,与双子叶及木本植物的亲缘较远。通过荧光定量分析,该基因在毛竹不同发育时期和不同组织中表达丰度不同,在冬笋中表达量最高,其次是春笋中部、春笋顶部、一年生叶、二年生茎、三年生茎、杆箨、一年生根、一年生茎、春笋基部、春笋。通过对C4H,CCoAOMT1,CCoAOMT2,4CL在毛竹不同组织中的表达量的分析,结果证明这四种基因与发育时期维管组织的细胞壁加厚相关。CCoAOMT1,CCoAOMT2的表达量明显高于C4H, 4CL,说明CCoAOMT1,CCoAOMT2基因在竹子快速生长时期需要大量表达。这4个基因在维管组织发育过程中的特异表达说明它们可作为调控竹子木质素合成的外源基因应用于竹子基因工程。更多还原

【Abstract】 The cDNA encoding full-length of CCoAOMT1, CCoAOMT2, C4H, 4CL genes were cloned from cDNA prepared from tissue culture seedings of Moso bamboo using RT-PCR and RACE methods. Its nucleotide sequences and the encoded amino acid sequences were analyzed. The important component to explicit the functions of gene is to carry out the study of gene expression with tissues,for instance, winter-shoot, whole spring-shoot, top-springshoot, mid-springshoot, base-shoot, leaf, leaf sheath, root, 1-year-old stem, 2-year-old stem and 3-year-old stem, using technology of fluorescence quantitative real-time PCR(QRT-PCR). To determine their expression properities and confirm their involvement in the lignin biosythesis in bamboo genes.(1)The whole open reading frame of CCoAOMT1 gene is 1045 bp encoding 262 amina acids. From the analysis of the sequence through the website of SMART and ExPASy against NCBI database, it is found that CCoAOMT1 is belong to p450 family and there are nine EGF-like domain signature 1, fifteen VWFC domain signature,one Integrins beta chain cysteine-rich domain signature, two Integrins beta chain cysteine-rich domain signature, two 2Fe-2S ferredoxins, iron-sulfur binding region signature, one 4Fe-2S ferredoxins, iron-sulfur binding region signature, one Anaphylatoxin domain signature, two Thiolases active site, four C-terminal cystine knot signatureone Insulin-like growth factor- binding protein (IGFBP) N-terminal domain signature.The amino acid sequence showed high similarity with the corresponding genes from monocots Bambusa oldhamii, oryza and zea mays at 99%, 88% and 88%,respectively. Expression analysis of CoAOMT1 by real time quantitative PCR showed the expression level in different tissues from high to low was in the following order: top-springshoot and mid-springshoot, base-shoot, leaf sheath, winter-shoot , 2-year-old stem, 1-year-old stem, root, whole spring-shoot, leaf, and the lowest expression level in the 3-year-old stem. (2)The whole open reading frame of CCoAOMT2 gene is 1131 bp encoding 262 amina acids. From the analysis of the sequence through the website of SMART and ExPASy, it is found that CCoAOMT2 is also belong to p450 family and there are nine EGF-like domain signature 1, fifteen VWFC domain signature, one Integrins beta chain cysteine-rich domain signature, four 2Fe-2S ferredoxins, iron-sulfur binding region signature, two 4Fe-2S ferredoxins, iron-sulfur binding region signature, three Anaphylatoxin domain signature, two Thiolases active site, three C-terminal cystine knot signature.The amino acid sequence showed high similarity with the corresponding genes from monocots oryza and zea mays at 88% and 87%,respectively. Expression analysis of CoAOMT2 by real time quantitative PCR showed the expression level in different tissues from high to low was in the following order: top-springshoot and mid-springshoot, winter-shoot, base-shoot, 2-year-old stem, 3-year-old stem, whole spring-shoot, and the lowest expression level in the leaf sheath, 1-year-old stem, root, leaf.(3)The whole open reading frame of C4H gene is 1006 bp encoding 502 amina acids. From the analysis of the sequence through the website of SMART and ExPASy, it is found that C4H is belong to p450 family and there are twelve EGF-like domain signature 1, two C-terminal cystine knot signature, twelve VWFC domain signature, one Integrins beta chain cysteine-rich domain signature, six 2Fe-2S ferredoxins, iron-sulfur binding region signature, one Thiolases active site, one 4Fe-2S ferredoxins, iron-sulfur binding region signature, one Mammalian defensins signature, three Anaphylatoxin domain signature.The amino acid sequence showed high similarity with the corresponding genes from monocots Sorghum Moench, oryza and zea mays at 88%, 88% and 87%,respectively. Expression analysis of C4H by real time quantitative PCR showed the expression level in different tissues from high to low was in the following order: winter-shoot, top-spring shoot, mid-spring shoot, leaf, 3-year-old stem, root, leaf sheath, 2-year-old stem, whole spring-shoot, 1-year-old stem, the base-shoot.(4) The whole open reading frame of 4CL gene is 1792 bp encoding 543 amina acids. From the analysis of the sequence through the website of SMART and ExPASy, it is found that 4CL is belong to AMP-binding family and there are ten EGF-like domain signature 1, two C-te rminal cystine knot signature, twenty VWFC domain signature, four Integrins beta chain cystei ne-rich domain signature, six 2Fe-2S ferredoxins, iron-sulfur binding region signature, two 4Fe-2S ferredoxins, iron-sulfur binding region signature, three Mammalian defensins signature, two Anaphylatoxin domain signature, two Insulin-like growth factor-binding protein (IGFBP) N-terminal domain signature, one Janus-faced atracotoxin (J-ACTX) family signature.The amino acid sequence showed high similarity with the corresponding genes from monocots Neosinocalamus affinis, oryza and zea mays at 100%, 97% and 83%,respectively. Expression analysis of 4CL by real time quantitative PCR showed the expression level in different tissues from high to low was in the following order: winter-shoot, mid-springshoot, top-springshoot, leaf, 2-year -old stem, 3-year-old stem, leaf sheath, root, 1-year-old stem, base-springshoot, the lowest expr ession level in the whole spring-shoot. This indicates that the cloned CCoAOMT1、CCoAOMT2、C4H、4CL involved in the cell wall deposition during the development of vascular tissues thus could be used in modulation of lignin biosynthesis in bamboo through genetic engineering.更多还原