竹笋细胞壁木质化及转录组差异基因表达分析与功能初探

发布时间：2018-11-28 19:42

【摘要】：竹笋即竹子膨大的芽和幼嫩的茎,具有高纤低脂,营养齐全的特点,备受消费者青睐。但竹笋采收后很快出现木质化,丧失鲜嫩可口的食用品质,成为竹笋产业发展的重要限制因素。因此,探究竹笋木质化的发生及其分子机理为进一步研究延长竹笋贮运期、降低采后损耗的新技术提供了理论支撑。本论文以毛竹笋上、中、下3个部位为试验材料,研究了毛竹笋不同部位细胞壁的成分变化以及与木质化有关的转录组差异基因的表达情况,并初步分析了半纤维素木聚糖合成的关键基因PeIRX10在竹笋组织次生细胞壁形成中的功能。得到如下结论:(1)毛竹笋不同部位因木质化程度不同,细胞壁中各组分的含量不同。沿着竹笋的生长方向,木质素和纤维素含量由上部到下部逐渐增加,而果胶和半纤维素的含量逐渐降低,同时木质素合成相关酶PAL、CAD和C4H的活性也由上部到下部逐渐增加,说明毛竹笋在下部的木质化程度高于上部和中部。(2)对毛竹笋的上、中、下三个部分分别进行RNA-seq分析,共获得了大约133.9百万条reads,总碱基数为18.13Gbp。在上vs中、上vs下和中vs下的两两比对中,发现的DEG数目分别为3840、3518和3925,并对其进行功能注释。然后筛选并分析了木质化过程中与细胞壁代谢相关的差异基因,得到了编码纤维素、半纤维素和木质素合成途径中相关酶基因的表达特点,并对其进行qRT-PCR验证,qRT-PCR结果与RPKM值一致。与纤维素和木质素合成相关的基因在下部和中部的表达量高于上部,这些基因在中下部表达量的上调与其较高的木质化程度有关。(3)构建了木聚糖合成关键基因PeIRX10过量表达载体并转化模式植物拟南芥,发现PeIRX10基因的过量表达在拟南芥irx1(-/-)irx10(-/-)双突植株中可以使其表型恢复正常、细胞壁不正常的木质部消失,且细胞壁中木聚糖含量恢复到正常水平。表明毛竹的PeIRX10基因很可能与拟南芥AtIRX10基因具有相同的生物学功能,通过参与木聚糖的生物合成而导致竹笋组织次生细胞壁的形成。
[Abstract]:Bamboo shoots are bamboo shoots and young stems with high fiber and low fat and complete nutrition, which are favored by consumers. However, the lignification of bamboo shoots soon after harvest, the loss of fresh and delicious edible quality, has become an important limiting factor for the development of bamboo shoot industry. Therefore, the study of the lignification of bamboo shoots and its molecular mechanism provide theoretical support for further study on the new technology of prolonging the storage and transportation period of bamboo shoots and reducing post-harvest loss. In this paper, the composition changes of cell wall and the expression of transcriptional differentially expressed genes in different parts of bamboo shoot were studied. The function of PeIRX10, the key gene of hemicellulose xylan synthesis, in the formation of secondary cell wall of bamboo shoot was analyzed. The conclusions are as follows: (1) the content of components in the cell wall of bamboo shoots is different because of the different degree of lignification. Along the growth direction of bamboo shoots, the content of lignin and cellulose increased gradually from the top to the bottom, while the contents of pectin and hemicellulose decreased gradually, and the activities of lignin synthase PAL,CAD and C4H increased gradually from the upper to the lower. The results showed that the lignification degree of bamboo shoots in the lower part was higher than that in the upper part and the middle part. (2) the total base number of 133.9 million reads, was obtained by RNA-seq analysis of the upper, middle and lower parts of the bamboo shoot. In the pairwise comparison of the upper vs, the upper vs and the middle vs, the number of DEG found is 3840m3518 and 3925, respectively, and its function is annotated. Then the differentially expressed genes related to cell wall metabolism during lignification were screened and analyzed. The expression characteristics of enzyme genes related to cellulosic hemicellulose and lignin biosynthesis were obtained and verified by qRT-PCR. QRT-PCR results are consistent with RPKM values. The genes related to cellulose and lignin synthesis expressed more in the lower and middle parts than in the upper ones. The upregulation of these genes in the middle and lower part is related to the higher lignification degree. (3) the overexpression vector of PeIRX10, the key gene of xylan synthesis, was constructed and transformed into Arabidopsis thaliana. It was found that overexpression of PeIRX10 gene in Arabidopsis thaliana irx1 (- / -) irx10 (- / -) could make the phenotype return to normal, the abnormal xylem disappeared, and the xylan content in the cell wall returned to normal level. It was suggested that the PeIRX10 gene of Phyllostachys pubescens had the same biological function as the AtIRX10 gene of Arabidopsis thaliana, which resulted in the formation of secondary cell wall of bamboo shoot tissue by taking part in the biosynthesis of xylan.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

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