条斑紫菜抗性相关代谢路径分析及其SSR分子标记筛选

发布时间：2018-06-02 22:03

本文选题：条斑紫菜 + 非生物胁迫　；参考：《中国科学院研究生院(海洋研究所)》2016年博士论文

【摘要】：条斑紫菜是一种重要的经济海藻。近年来条斑紫菜种质退化,影响了紫菜产业的发展,培育优良的种质成为亟需解决的问题。鉴于条斑紫菜生活环境的特点,可以从提高其抗性方面进行良种培育。良种培育需要有优良性状的种质资源,但是条斑紫菜育种研究相对陆地作物来说起步较晚,基础研究也比较薄弱,目前还没有明确的与抗性特征相关的位点和分子标记。正因如此,本论文聚焦条斑紫菜的抗性育种,一方面研究其光合作用过程对盐胁迫条件的响应,确定藻体抗性特征的关键位点;另一方面基于条斑紫菜基因组测序信息开发SSR标记,通过人工诱变扩展其遗传多样性,为定位抗性特征相关数量性状位点奠定基础。主要研究结果如下:(1)分析条斑紫菜光合作用过程对盐胁迫的响应,发现光系统II(PSII)对高盐胁迫条件比光系统I(PSI)更敏感。在高盐(120‰)条件下,PSII的实际光合量子产量(effective PSII quantum yield,YII)几乎为零,但是PSI的实际光合量子产量(effective PSI quantum yield,YI)维持较高活性,表明在PSII被抑制时PSI运转时的电子来源于基质侧还原力。条斑紫菜随着盐度的升高藻体淀粉含量呈下降的趋势,可溶性糖含量却与其相反;同时,随着盐度的升高磷酸戊糖途径(oxidative pentose phosphate pathway,OPPP)的关键酶6-磷酸葡萄糖脱氢酶(glucose-6-phosphate dehydrogenase,EC 1.1.1.49,G6PDH)和6-磷酸葡糖酸脱氢酶(6-phosphogluconate dehydrogenase,EC 1.1.1.44,6PGDH)的活性呈上升趋势,糖酵解途径中的重要酶3-磷酸甘油醛脱氢酶(cytosolic glyceraldehyde 3-phosphate dehydrogenase,EC 1.2.1.12,GAPDH)活性却呈下降的趋势。紫菜细胞还原力含量测定结果显示,由OPPP途径产生的NADPH含量呈升高的趋势,而糖酵解途径中产生的NADH含量却呈下降趋势。研究结果说明,在盐胁迫的条件下淀粉降解速度加快,产生的可溶性糖进入OPPP途径,产生的还原力NADPH推动了PSI的环式电子传递运行。(2)基于条斑紫菜全基因组测序信息,开发了700对完全重复类型的SSR标记。在270 M基因组中存在254495个SSR标记,平均每隔1 kb存在1个SSR标记;条斑紫菜的SSR标记包括2-6个碱基的重复,其中两碱基所占的比例最多,其次是三碱基。重复类型中CG/GC比例最高,其次是CGG/CCG类型。在保存的16个条斑紫菜样品中分析了SSR标记的多态性情况,发现98对SSR标记在这些样品中具有多态性。根据PCR产物电泳结果进行遗传分析,发现平均等位基因数(Na)为2.479;有效等位基因数(Ne)为1.558;香农信息指数(I)为0.543;观察杂合度(Ho)为0.168,期望杂合度(He)为0.32,PIC为0.35。聚类分析将这16个样品聚成了两大类,说明种质混杂,样品间存在基因交流。(3)化学(EMS)和物理(重离子束辐照)方法对条斑紫菜丝状体进行了诱变,结果表明:0.2 M EMS处理2 h、0.4 M EMS处理1.5 h和0.6 M EMS处理1h是比较有效的化学诱变方法;重离子束辐照方法表明100-150戈瑞(GY)是比较有效的剂量。随机选取20个重离子束辐照丝状体样品,用SSR分子标记分析其遗传多样性,结果表明:平均等位基因数为(Na)2.842;有效等位基因数(Ne)为1.935;香农信息指数(I)为0.756;观察杂合度(Ho)为0.656,期望杂合度(He)为0.46,PIC平均为0.39。诱变条斑紫菜聚类分析结果表明不同的样品基本按照重离子束辐照时所用的剂量进行聚类,这些样品被分成了几个亚类,最终这些亚类同对照聚到一起。这些诱变的样品较保存的条斑紫菜遗传多样性高。
[Abstract]:Porphyra yezoensis is an important economic seaweed. In recent years, the germplasm degradation of Porphyra yezoensis has affected the development of the Porphyra yezoensis industry. It is an urgent problem to cultivate fine germplasm. In view of the characteristics of the living environment of Porphyra yezoensis, it can be cultivated from the improvement of its resistance. The breeding research of Porphyra yezoensis is relatively late and the basic research is relatively weak. There are no specific loci and molecular markers related to resistance characteristics. This paper focuses on the resistance breeding of Porphyra yezoensis. On the one hand, it studies the response of the light cooperation process to the salt stress conditions and determines the resistance of the algae. On the other hand, based on the genome sequencing information of Porphyra yezoensis, SSR markers were developed to expand the genetic diversity by artificial mutagenesis to lay the foundation for the quantitative trait loci of the resistance characteristics. The main results are as follows: (1) the response of light cooperative process to salt stress of Porphyra yezoensis was analyzed, and the light system II (PS) was found. II) the high salt stress condition is more sensitive than the light system I (PSI). Under the condition of high salt (120 per thousand), the actual photosynthetic quantum yield of PSII (effective PSII quantum yield, YII) is almost zero, but the actual photosynthetic quantum yield of PSI (effective PSI) is highly active. With the increase of salinity, the starch content of the algae declined with the increase of salinity, but the content of soluble sugar was opposite. At the same time, with the increase of salinity, the key enzyme of pentose phosphate pathway (oxidative pentose phosphate pathway, OPPP) was 6- phosphoric glucose dehydrogenase (glucose-6-phosphate dehydrogenase, EC 1.1.1.49, G6P) DH) and 6- phosphate dehydrogenase (6-phosphogluconate dehydrogenase, EC 1.1.1.44,6PGDH) are on the rise. The activity of 3- phosphate glyceraldehyde dehydrogenase (cytosolic glyceraldehyde 3-phosphate dehydrogenase, EC) is declining. The results showed that the content of NADPH produced by OPPP pathway increased and the content of NADH produced in the glycolysis pathway decreased. The results showed that the degradation rate of starch was accelerated under salt stress, the soluble sugar produced into the OPPP pathway, and the resulting reducing force NADPH promoted the ring type electron transfer operation of PSI. (2) based on the results, the results showed that the degradation rate of soluble sugar produced by the glycolysis was accelerated. The whole genome sequencing information of Porphyra yezoensis has developed 700 pairs of fully duplicated SSR markers. There are 254495 SSR markers in the 270 M genome, with an average of 1 SSR markers per 1 KB; the SSR marker of Porphyra yezoensis includes 2-6 base repeats, among which two bases occupy the largest proportion, followed by the three base. The CG/GC ratio in the repeat type. The highest, followed by the CGG/CCG type. The polymorphism of the SSR markers was analyzed in the 16 preserved Porphyra samples, and 98 pairs of SSR markers were found to be polymorphic in these samples. The average number of alleles (Na) was 2.479, the number of effective alleles (Ne) was 1.558, and the Shannon information index (I) was found. ) for 0.543, the observation heterozygosity (Ho) was 0.168, the expected heterozygosity (He) was 0.32, and PIC was 0.35. clustering analysis to gather the 16 samples into two categories, indicating that the germplasm was mixed and the gene exchange existed between the samples. (3) chemical (EMS) and Physics (heavy ion beam irradiation) were used to mutagenate the filamentous body of Porphyra yezoensis. The results showed that 0.2 M EMS treatment 2 h, 0.4 M E. MS processing 1.5 h and 0.6 M EMS to treat 1H is a more effective chemical mutagenesis method; heavy ion beam irradiation method shows that 100-150 Gore (GY) is a more effective dose. 20 heavy ion beams are randomly selected to irradiate filamentous samples and the genetic diversity is analyzed with SSR molecular markers. The results show that the average allele number is (Na) 2.842, and the effective allele is effective. The factor (Ne) was 1.935, the Shannon information index (I) was 0.756, the observation heterozygosity (Ho) was 0.656, the expected heterozygosity (He) was 0.46, and the PIC was 0.39. mutagenesis. The cluster analysis of Porphyra yezoensis showed that the different samples were basically clustered according to the dose used in the heavy ion beam irradiation, and these samples were divided into several subclasses, and finally these subcategories were similar. Compared with the preserved Porphyra yezoensis, these mutagenic samples had higher genetic diversity.
【学位授予单位】：中国科学院研究生院(海洋研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S917.3

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