实验海洋生物学重点实验室知识库

    经济红藻—龙须菜已在我国南、北方进行了推广和养殖，显示出较强的经济、生态效益。龙须菜主要用于琼胶制备和鲍养殖的活鲜饵料。近年来，经过遗传选育，我们获得了绿色突变型龙须菜，通过养殖试验，我们发现其养殖性状保持稳定。本研究选择红色龙须菜（野生型）和绿色龙须菜（突变型），开展种质遗传分析，结合温度、盐度的影响，对其温度和盐度耐受响应进行分析，结果证实绿色龙须菜对高温、低盐均有较好的耐受性，本研究为龙须菜品种选育提供参考和依据。

    利用SSR标记开展种质鉴定分析，使用500个SSR标记对两种颜色龙须菜进行遗传分析，尚未筛选出绿色龙须菜区别于红色龙须菜的特异遗传标记。对两种颜色龙须菜的光合辅助色素藻胆蛋白基因进行测序发现其基因序列无差异。 两种颜色龙须菜在三种温度（8、20和30 °C）下培养7天，通过对比分析发现，30 °C处理后，红色龙须菜快速叶绿素荧光诱导动力学OJIP曲线显著下降，而绿色龙须菜的OJIP曲线受高温影响较小；绿色龙须菜光性能指数（PI_abs）在30 °C时下降幅度小于红色龙须菜，且高温胁迫后绿色龙须菜有较强的过氧化物酶（POD）和过氧化氢酶（CAT）酶活；30 °C处理后，绿色龙须菜Hsp70和Hsp90的转录水平高于红色龙须菜。8 °C处理后，两种颜色龙须菜的光合性能均受到严重抑制，但绿色龙须菜的最大光化学效率（F_V/Fm）和PI_abs下降幅度均高于红色龙须菜，表明其光合性能受到了更严重的抑制。在8 °C下，绿色龙须菜超氧化物歧化酶（SOD）活力上升，其Hsp70和Hsp90高水平转录。

    两种颜色龙须菜在低盐条件（盐度28和22）下培养3天，我们对其叶绿素荧光进行了对比分析。在低盐28处理下，红色龙须菜的光合性能明显下降，最大相对电子传递速率（rETR_max）下降了20.4%，而绿色龙须菜无明显变化。在低盐22处理下，红色龙须菜的Fv/Fm下降了16.0%，而绿色龙须菜下降了11.9%；红色龙须菜电子传递量子产额（φEo）下降了20.3%，而绿色龙须菜无显著变化；在盐度22下，绿色龙须菜光能利用效率（α）和rETR_max的下降幅度均小于红色龙须菜。

    The commercial red seaweed, Gracilariopsis lemaneiformis, has been widely applied for large-scale cultivation in the south and north of China, and exhibited obvious economic profits and ecological value. Usually, G. lemaneiformis was mainly used for manufacturing agar and used as the fresh feeder for abalone cultivation. In recent years, through the selection and culture trials, we obtained one green mutant of G. lemaneiformis and proved its availability in aquaculture. Here in this study, the red type (the wild type) and the green type (the mutant) of G. lemaneiformis were used for the characterization of genetic and tolerance features to temperature and salinity. It was proved that the green type could be tolerant to high temperature and low salinity, and our yielded data could provide parameters for the selection and application of G. lemaneiformis on large scale in the future.

    Using SSR markers to carry out germplasm identification, 500 SSR markers were used to carry out genetic analysis of two types of G. lemaneiformis, and the specific genetic markers that distinguish the green type from the red type could not be screened. The phycobiliprotein gene of photosynthetic auxiliary pigment of two types of G. lemaneiformis was sequenced, and it was found that there was no difference in the gene sequence.

    To culture the red and green types of G. lemaneiformis under 8, 20, and 30 °C respectively for 7 days, we found the fast chlorophyll fluorescence intensity(OJIP) decreased at 30 °C in the red type, while no significant change was observed in the green type. At 30 °C, the decrease of the performance index (PI_abs) in the green type was less than that in the red type, and stronger peroxidase (POD) and catalase (CAT) activities were measured in the green type. Transcriptions of Hsp70 and Hsp90 in the green type were higher than that in the red type at 30 °C. Under 8 °C, the photosynthetic performance of the two G. lemaneiformis types significantly decreased, but the decreased ratios of maximum quantum yield for primary photochemistry (F_V/Fm) and PI_abs in the green type were higher than that of the red type, which reflected its photosynthetic activity was more seriously inhibited. At 8 °C, the superoxide dismutase (SOD) activity and transcriptions of Hsp70 and Hsp90 were increased in the green type.

    In the comparison of 28 and 22 salinities cultured to the green and red types of G. lemaneiformis for 3 days, it was found that the photosynthetic activity in the red type significantly decreased with 20.4% degrees of maximum relative electron transfer rate (rETR_max) at 28 salinity, and no significant change was observed in the green type. Under the 22 salinity condition, the Fv/Fm value decreased by 16.0% and 11.9% respectively in the red and green types, and the value of quantum yield for electron transport (φEo) decreased by 20.3% in the red type at 22 salinity, but no obvious change was observed in the green type. Compared with the red type, the decreases in light energy use efficiency (α) and rETR_max in the green type were lower under 22 salinity.

第1章 引言.... 1

1.1 龙须菜生物学特征及研究背景... 1

1.1.1 龙须菜生物学特征... 1

1.1.2 龙须菜的应用... 2

1.1.3 龙须菜遗传选育研究进展... 3

1.2 龙须菜种质鉴定分析... 4

1.3 海藻对环境胁迫的响应... 4

1.3.1 海藻对温度的生理响应... 4

1.3.2 海藻对盐度的生理响应... 5

1.3.3 海藻对光照的生理响应... 5

1.3.4 海藻对CO₂的生理响应... 6

1.3.5 海藻对营养盐的生理响应... 6

1.3.6 海藻对重金属的生理响应... 6

1.4 海藻光合作用及检测分析... 7

1.4.1 光合作用光反应基本原理... 7

1.4.2 叶绿素荧光动力学曲线... 9

1.4.3 快速光响应曲线... 11

1.5 海藻活性氧与抗氧化酶... 11

1.6 热休克蛋白... 12

1.7 研究目的及意义... 12

第2章 两种颜色龙须菜种质遗传分析.... 13

2.1 材料与方法... 13

2.1.1 实验材料... 13

2.1.2 实验仪器... 13

2.1.3 实验试剂... 13

2.1.4 实验方法... 14

2.2 实验结果... 17

2.2.1 SSR标记的开发... 17

2.2.2 藻红蛋白γ亚基扩增分析... 19

2.3 小结... 19

第3章 两种颜色龙须菜荧光比较.... 21

3.1 材料与方法... 21

3.1.1 实验材料... 21

3.1.2 实验仪器... 21

3.1.3 实验方法... 21

3.2 实验结果... 22

3.2.1 快速光响应曲线对比分析... 22

3.2.2 快速叶绿素荧光曲线对比分析... 23

3.2.3 叶绿素荧光参数对比分析... 24

3.2.4 高光谱反射曲线对比分析... 25

3.3 小结... 26

第4 章 温度对两种颜色龙须菜的影响研究.... 27

4.1 材料与方法... 27

4.1.1 实验材料... 27

4.1.2 实验仪器... 27

4.1.3 实验试剂... 28

4.1.4 实验方法... 28

4.2 实验结果... 36

4.2.1 温度胁迫后OJIP曲线对比分析... 36

4.2.2 温度胁迫后JIP-test雷达图对比分析... 37

4.2.3 温度胁迫后NPQ对比分析... 39

4.2.4 温度胁迫后OFR和MDA对比分析... 39

4.2.5 温度胁迫后抗氧化酶活对比分析... 40

4.2.6 温度胁迫后Hsp荧光定量对比分析... 41

4.3 小结... 42

4.3.1 高温胁迫... 42

4.3.2 低温胁迫... 43

第5章 低盐对两种颜色龙须菜的影响研究.... 45

5.1 材料与方法... 45

5.1.1 实验材料... 45

5.1.2 实验仪器... 45

5.1.3 实验方法... 45

5.2 实验结果... 46

5.2.1 低盐胁迫后快速光响应曲线对比分析... 46

5.2.2 低盐胁迫后叶绿素荧光参数对比分析... 47

5.3 小结... 49

第6章 结论.... 51

参考文献.... 53

附 录.... 67

致 谢.... 101

作者简历及攻读学位期间发表的学术论文与研究成果.... 103