|关键词||条斑紫菜 非生物胁迫 磷酸戊糖途径 G6pdh Ssr 抗性筛选|
（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 18.104.22.168, G6PDH）和6-磷酸葡糖酸脱氢酶（6-phosphogluconate dehydrogenase, EC 22.214.171.124, 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 126.96.36.199, 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1 h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。
Porphyra yezoensis (P. yezoensis ) is one of the most important economically marine algae. In recent years, the quality of the P. yesoensis is reduced, which greatly affects the development of seaweed industry. The cultivation of best quality algae with stress resistance becomes the urgent need. Germplasm resource of high quality is the key point of breeding stress resistance species. However, the research on the breeding of marine algae started late, and the basic research of it is weak. At present, there is no physiological and molecular markers to screen the excellent germplasm resources. Therefore, it is important to find the key position of P. yesoensis in response to abiotic stress and develop the molecular markers. In this study, we focused on resistant breeding of P. yezoensis. More attentions were paid to determine the key position of resistance characteristics under salt stress in P. yezoensis and develop SSR markers for resistance characteristics of quantitative trait loci. In addition, the genetic diversity was extended by artificial mutation, all of which laid the foundation for the quantitative trait loci of the resistance traits. These results mainly include the follows:
(1) The photosynthetic (PS) responses of P. yezoensis blades under salt stress were studied. Our results showed that when the effective photochemical quantum yield of PSII almost decreased to zero under high salt stress, while YI still had a relative high activity rate. PSII is therefore more sensitive to salt stress than PSI. These results demonstrated that under severe salt stress (120‰ salinity), the electro source of PSI was derived from a source other than PSII, and that stromal reductants probably supported the operation of cycle electron flow around PSI. Under salt stress, the starch content decreased and the soluble sugar levels increased. The G6PDH and 6PGDH (EC 188.8.131.52) activities increased, but the GAPDH activity decreased. Furthermore, the NADPH content increased, and NADH decreased, which suggested that soluble sugar entered the oxidative pentose phosphate pathway (OPPP). All these results suggested that NADPH from OPPP increases the cyclic electron flow around PSI in high intertidal macroalgae under severe salt stress.
(2) Based on the sequenced genomic, 700 SSR markers of complete repeat were developed. There are 254495 SSR sites in the 270 M genomic, with one SSR marker every 1 kb on average. These results showed that the SSR markers were abundant, including all the 2-6 bases SSR makers. Among the genomic two base repeats have the largest proportion, followed by three bases. The highest proportion of repeat types is CG/GC type, followed by CGG/CCG type. The polymorphism of 700 SSR markers was analyzed in 16 P. yezoensis conchocelis from different places. The results demonstrated that 98 SSR markers were polymorphic in these samples. Genetic analysis was carried out on the polymorphic makers according to the PCR results. By GenALEx6.3 software: The average value of alleles was 2.479, effective alleles (Ne) was 1.558, diversity information index (I) was 0.543, observed heterozygosity (Ho) was 0.168, expected heterozygosity (He) was 0.32, PIC was 0.35. Cluster analysis was carried out by UPGMA method. The results showed that these 16 conchocelis of P. yezoensis were clustered into two main branches. All these results suggested that the gerplasm of P. yezoensis have been mixed, and there is gene flow exchange between them.
(3) In this study, the EMS mutation and heavy ion beam were used to get the mutant strains of P. yezoensis conchocelis. Research shows that the treatment of 0.2 M EMS concentration for 2 h, 0.4 M EMS concentration for 1.5 h and 0.6 M EMS concentration for 1 h are effective ways to get P. yezoensis mutant strains. Which provides a reference for other uses of EMS induction in P. yezoensis. Heavy ion beam irradiation method showed that the 100-150 GY dose was effective dose. Twenty conchocelis were randomly selected from the mutant strains, then the genetic diversity was analyzed by SSR markers. The average value of alleles (Na) was 2.842, effective alleles (Ne) was 1.935, diversity information index (I) was 0.756, observed heterozygosity (Ho) was 0.656, expected heterozygosity (He) was 0.464, and the PIC was 0.39. The samples were clustered approximately in accordance with the dose of heavy ion beam, and divided into several sub categories which were clustered with the control conchocelis. The genetic diversity of these mutant strains is more abundant then that of the16 P. yezoensis conchocelis. overall, the mutagenic method can enrich the genetic diversity of P. yezoensis.
|鲁晓萍. 条斑紫菜抗性相关代谢路径分析及SSR分子标记筛选[D]. 北京. 中国科学院大学,2016.|