【作者】 陶章生；

【导师】 彭良才； 丰胜求；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 拟南芥是重要模式植物,已发现10个纤维素合酶基因(AtCesA1～AtCesA10)。AtCESA1、AtCESA3、AtCESA6是初生壁纤维素合酶基因,AtCESA4、AtCESA7、AtCESA8是次生壁纤维素合酶基因,分别参与拟南芥不同生育期的纤维素合成；根据突变体的表型变化推测AtCESA2、AtCESA5、AtCESA9对AtCESA6可能存在部分功能冗余。但AtCESA2、AtCESA5对AtCESA6的冗余过程,AtCESA2、AtCESA5在纤维素合成中的作用等问题还不清楚。本实验根据拟南芥8个纤维素合酶基因(AtCesA1～AtCesA8)序列,在高变区Ⅰ处设计引物,合成了GST-AtCESAs融合蛋白,制备了抗体。以AtCesA6突变体为材料(prc1-1、cesa6),从基因水平、蛋白水平和细胞壁成分三方面研究了在光暗条件下AtCESA2和AtCESA5对AtCESA6的冗余功能,探讨AtCESA2、AtCES-A5在纤维素合成中的作用。主要结果如下：1.纤维素合酶抗体的制备和检测制备了AtCESA1～AtCESA8的多克隆抗体。Western-blotting检测发现：AtCESA4和AtCESA7有严重的交叉反应,所以这两个抗体不能使用；其它抗体都能在拟南芥原生质膜上免疫出相应的特异条带。2.光暗处理下拟南芥下胚轴AtCesAs的表达分析GUS染色结果和Real-time PCR结果表明：AtCesA1、AtCesA3、AtCesA6暗处理下表达明显增加；AtCesA2暗处理下表达量较低,而暗转光后表达量明显增加；AtCesA5光暗处理下表达变化趋势不显著。3.光暗处理下拟南芥突变体纤维素合酶基因水平分析与野生型相比,突变体prc1-1、cesa6光暗处理下的AtCesAs的表达都显著降低；光暗比较发现：暗处理下prc1-1、cesa6的AtCesA1、AtCesA3、AtCesA5的表达明显高于光处理。4.光暗处理下拟南芥突变体纤维素合酶蛋白水平分析与野生型相比,光暗处理下prc1-1、cesa6原生质膜总蛋白中的AtCESA6显著降低,AtCESA5显著增加。光下AtCESA2在AtCESAs中所占的比例并没有明显的变化,而暗处理下AtCESA2在AtCESAs中所占比例显著降低。因此推测,AtCESA5在光暗9天的prc1-1、cesa6中都能替补AtCESA6的部分功能,维持突变体存活；AtCESA2只在光下9天的prc1-1、cesa6中弥补AtCESA6的部分功能,使植株能够正常生长。但是在突变体纤维素合酶复合体中AtCESA1、AtCESA3、AtCESA6、AtCESA2、AtCESA5的含量都降低了。与野生型相比,光下突变体prc1-1、cesa6的纤维素合成量显著增加,胼胝质合成量没有明显变化,cesa6的更为显著；而暗处理下纤维素、胼胝质合成量显著降低,prc1-1的更为明显。5.拟南芥突变体成分分析与野生型相比,光下9天,prc1-1的晶体纤维素显著下降,非晶体纤维素显著增加；cesa6的晶体纤维素含量降低了,非晶体纤维素、半纤维素、果胶质、可溶性糖都显著增加。暗处理9天,prc1-1的晶体纤维素显著下降,非晶体纤维素、半纤维素、果胶质、可溶性糖都显著增加；cesa6的晶体纤维素显著下降,非晶体纤维素没有明显变化,而半纤维素、果胶质、可溶性糖都显著增加。6. AtCesA6突变后对次生壁的影响与野生型相比,prc1-1、cesa6秸秆的果胶质显著增加,半纤维素、非晶体纤维素和晶体纤维素都显著降低,木质素变化不显著。prc1-1、cesa6秸秆纤维素的聚合度、结晶度没有明显的变化；半纤维素的支链有明显的增多；木质素单体的H显著增加,G、S显著降低。与野生型相比,cesa6的纤维素酶酶解产糖率最高增加(8.83±0.87)%,prcl-1增加(5.68±0.49)%；而IRX-3的增加了(36.63±2.67)%。更多还原

【Abstract】 At least 10 CESA sequences (AtCesA1～AtCesA10) have been identified in Arabid-opsis to involve in cellulose synthesis. AtCESA1, AtCESA3 and AtCESA6 are characterized for primary cell wall synthesis, whereas AtCESA4, AtCESA7 and AtCESA8 are for secondary cell wall. It has been reported that AtCESA2, AtCESA5 and AtCESA9 might be partial functional redundancy with AtCESA6. However, it is not clear about the redundancy mechanism for cellulose biosynthesis in plant.In this study, we amplified cDNA fragments of AtCesAs variable region by RT-PCR, isolated GST-AtCESAs fusion proteins, and prepared their antibodies. We observed gene expression, detected proteins and analyze CESA activities in vitro using AtCesA6 mutant (prc1-1, cesa6).Major results were described below:1. Preparation and detection of AtCesAs AntibodiesWe prepared their (AtCesA1～AtCesA8) antibodies. Western-blotting analysis indicat-ed that the antibodies (Anti-AtCESA1, Anti-AtCESA3, Anti-AtCESA6, Anti-AtCESA2, Anti-AtCESA5, Anti-AtCESA8) showed specific bands against proteins extracted from the plasma membrane fraction in Arabidopsis. But, I found cross-reaction bands in Anti-AtCESA4 and Anti-AtCESA7.2. Expression observation of AtCesAs in hypocotyls under light/dark treatmentsGUS staining and the Real-time PCR showed that AtCesA1, AtCesA3 and AtCesA6 expression levels were significantly increased under dark treatment. AtCesA2 showed a decreased expression under dark and was remarkable increased under ligh. AtCesA5 was little changed under both treatments.3. Expression analysis of AtCesAs in mutantsCompared with wild type, AtCesAs in mutants were decreased remarkably under 9-day-old light/dark conditions. Under dark treatment, AtCesA1, AtCesA3 and AtCesA5 in mutants were expressed much higher than ones under light.4. Test of AtCESA proteins and analysis of AtCESA activity in vitroAtCESA6 protein was reduced whereas AtCESA5 was raised in the plasma membrane fractions of mutants (prc1-1, cesa6) under light/dark treatments, compared with wild type. AtCESA2 showed no difference under light, but was reduced under dark. It suggested that AtCESA5 may partially replace AtCESA6 for the survival of mutants under both light and dark treatments, while AtCESA2 can only play a partial substite role under light condition. However, amounts of the complex composed of AtCESAl, AtCESA3, and AtCESA6/AtCESA2/AtCESA5 in mutants were much less than ones in wild type.Cellulose synthesis in vitro indicated thatβ-1,4-glucan product was much higher thanβ-1,3-glucan in mutants and wild type under light condition, but bothβ-1,4-glucan andβ-1,3-glucan were remarably decreased under dark condition, especially for prc1-1 mutant.5. Determination of cell wall compositionUnder light treatment, mutants(prc1-1 & cesa6) were determined with much less crystalline cellulose and more non-crystalline cellulose than wild type, and mutant (cesa6) showed additionally increased hemicellulose, pectin and soluble sugar. As a contrast, mutant prc1-1 under dark was decreased in crystalline cellulose, but increased in non-crystalline cellulose, hemicelluloses, pectins and soluble sugar. Mutant cesa6 showed a decrease in crystalline cellulose and an increase in hemicellulose, pectins and soluble sugars.6. The effect of secondary cell wall caused by the mutation of AtCesA6Compared with wild type, prc1-1 and cesa6 were determined with much less hemi-cellulose, non-crystalline cellulose, crystalline cellulose and more pectin, but showed no difference in lignin. The mutants(prc1-1 & cesa6) were increased in side chains of hemi-cellulose, H unit of lignin, but decreased in G and S units of lignin. However, DP and CrI of cellulose were much unchanged in mutants’ straw. Under the treatment of cellulase, the relased sugars in cesa6 and prc1-1 can be increased by 8.83±0.87% and 5.68±0.49%, respectively, and IRX-3 can be increased by 36.63±2.67%.更多还原