实验海洋生物学重点实验室知识库

条斑紫菜（Pyropia yezoensis）生长在潮间带海域，随着潮汐作用，周期性地经历干出失水与浸没复水的生境转换，其体内必然进化出了独特的抗逆机制，包括抗氧化机制。而脱落酸（ABA）作为一种抗逆激素，在生物体逆境响应中发挥重要作用。因此，探究ABA与抗氧化机制的作用对条斑紫菜抗逆机制的解析具有很高的科学研究价值。我们以条斑紫菜叶状体为实验材料，测定了不同处理条件下条斑紫菜光合参数、ABA含量及相关抗氧化酶活性的变化，探究条斑紫菜叶状体中ABA对抗氧化系统在应对逆境胁迫时发挥的具体调控作用。通过转录组数据，在分子水平上分析、验证ABA对相关抗氧化酶基因表达调控作用。同时，结合条斑紫菜生产中实际情况，探究条斑紫菜高温烂菜现象与藻体中抗氧化机制及ABA的作用。最后，根据转录组中发现的与活性氧（ROS）清除相关的关键基因PyMPV17，通过转基因技术获得PyMPV17过表达及沉默藻株，以验证相关抗逆代谢途径，为紫菜抗逆品系的培育奠定理论基础。主要研究结果有以下四点：

1. 高盐胁迫条件下，条斑紫菜ABA对抗氧化酶活性有正向调控作用。高盐胁迫条斑紫菜藻体，同时添加ABA合成抑制剂及外源ABA测定不同抗氧化酶活性及H₂O₂含量变化。结果显示，ABA抑制剂（Clo、Mev）处理条斑紫菜藻体，抗氧化酶活性（CAT、SOD、APX、GR）有下调趋势，但添加ABA后抑制作用减弱，甚至与不加抑制剂处理的对照相比，有显著上调的趋势。转录组数据显示，外源ABA处理藻体后，相关抗氧化酶基因表达量显著提高，与酶活数据趋势一致。同时，我们测定了H₂O₂含量变化，结果显示ABA降低了藻体中H₂O₂的含量。我们推测，胁迫条件下藻体中相关抗氧化酶发挥作用，将藻体中H₂O₂转化为H₂O，从而降低藻体中H₂O₂含量。

2. 条斑紫菜藻体中，ABA的前体IPP可能由两条途径-MVA途径及MEP途径同时合成，但主要通过MEP途径合成。条斑紫菜ABA的具体合成途径可能同高等植物相似，主要以类胡萝卜素途径合成。我们选择IPP两条途径的抑制剂同时处理藻体，测定ABA含量，结果发现ABA含量均显著降低。但转录组数据中显示，MEP途径中7个基因被注释到，包括DXS、DXR、MCT、CMK、MECPS、HDS、HDR，而MVA途径中只注释到AATC基因。且抑制剂处理藻体中只有MEP途径中的DXS酶基因表达显著降低，所以我们猜测藻类中ABA前体IPP合成主要通过MEP途径，但存在MVA途径。同时，我们的转录组数据中注释到多个ABA类胡萝卜素合成途径，包括FPS，ZEP，PDS，SDR等，其中SDR是差异表达基因，抑制剂添加能显著降低该基因表达量，所以条斑紫菜中ABA合成可能通过类胡萝卜素途径合成。

3. 研究高温胁迫及海水盐度降低条件下ABA介导的抗氧化酶及其转录水平变化，发现在条斑紫菜栽培过程中ABA与抗氧化系统发挥重要作用。通过测定不同温度（15℃，20℃）、不同盐度（30，25，20）条件下藻体中ABA含量及相关抗氧化酶活性及表达量的变化，结果显示，低盐20条件下，20℃的高温对条斑紫菜光合作用系统会造成一定损伤，虽然此条件下未检测到H₂O₂的累积，但丙二醛（MDA）含量在盐度降低的样本中显著上调。低盐20条件下培养样本在温度升高时超氧化物歧化酶（SOD）及过氧化物酶（POD）活性上调。然而，本文测定的其他所有抗氧化酶及相关基因的表达在盐度20、温度20℃条件下均明显下调，抗氧化酶表达的下调说明低盐高温条件下，藻体氧自由基清除能力受到抑制。因此我们认为，在条斑紫菜海区栽培生产中，因高温诱发的病烂很可能与ABA含量降低、抗氧化酶系统的抑制直接相关，这可能是生产过程中，紫菜高温病烂发生的生理基础之一。

4. 根据转录组数据，发现PyMPV17基因在不同处理条件下差异表达。基于ABA介导抗氧化酶上调的可能机制及在多种逆境条件下的功能研究，筛选获得关键基因PyMPV17，通过转基因技术分别获得过表达及沉默藻株，并进行DNA及RNA水平的验证。下一步我们将进行蛋白水平验证、抗逆表型及相关基因表达的研究，以验证目标基因功能及在条斑紫菜新品系培育中的应用价值。

Pyropia yezoensis(Py. yezoensis) grows in the intertidal zone. With the tide, it periodically undergoes the habitat conversion of dehydration and rehydration. Therefore, it was evolved a unique stress resistance mechanism, including antioxidative system. Additionally, abscisic acid (ABA) plays an important role in responding to stress. Therefore, it has a high scientific research value for the analysis of algae stress resistance mechanism. Consequently, we used the Py. yezoensis thallus as the experimental material, the changes of photosynthetic parameters, ABA content and related antioxidant enzyme activities was measured under different treatment conditions, and explored the specific regulatory mechanism of stress hormone ABA and antioxidant system under high salinity stress in Py. yezoensis. At the same time, transcriptome data were analyzed and verified the specific regulatory mechanism of ABA on major antioxidant enzymes at the molecular level. On the other hand, combined with the actual production situation, explored the mechanism of rotten diseases induced by low salinity and high temperature and the role of ABA in Py. yezoensis thallus. Finally, we found a key gene PyMPV17 related to the removal of reactive oxygen species (ROS) in transcriptome data. And constructed the overexpression and silencing of PyMPV17 by transgenic technology to verify the related stress resistant metabolic pathway, it laid a theoretical foundation for the cultivation of stress resistant strains of Py.yezoensis. The main results are as follows:

1. Under high salinity stress, ABA has a positive regulation on antioxidant enzymes in Py. yezoensis. The activities of different antioxidant enzymes and the change of H₂O₂ content were determined by adding ABA synthesis inhibitors and exogenous ABA under hypersaline. The results show that the activities of related antioxidant enzymes(CAT, SOD, APX, GR) of Py. yezoensis treated with ABA inhibitors (Clo, Mev) had a downward trend, but the inhibition was weakened or even significantly increased after adding ABA. Transcriptome data showed that the expression of antioxidant enzyme genes were significantly increased after exogenous ABA treatment, which was consistent with the enzyme activity data. At the same time, we measured the change of H₂O₂ content. The results showed that ABA reduced the content of H₂O₂, It may be relevant that antioxidant enzymes play a significant role in transforming H₂O₂ to H₂O under ABA treatment, thus reducing the content of H₂O₂ in Py.yezoensis.

2. The precursor IPP of ABA may be synthesized by MVA pathway and MEP pathway simultaneously in Py.yezoensis, but mainly by MEP pathway. The specific synthesis pathway of ABA may be similar to higher plants, mainly by carotenoid pathway, also called indirect synthesis pathway. We selected inhibitors of the two pathways of IPP to treat the algae at the same time, and measured the ABA content. The results showed that the content of ABA decreased significantly. Meanwhile, transcriptome data showed that seven genes in MEP pathway were annotated, including DXS、DXR、MCT、CMK、MECPS、HDS、HDR. While only AATC gene was annotated in MVA pathway. In addition, only DXS gene expression in MEP pathway was significantly decreased in algae treated with inhibitors. Therefore, we speculated that the synthesis of ABA precursor IPP in algae was mainly through MEP pathway, but there was MVA pathway. At the same time, our transcriptome data annotated a number of ABA carotenoid synthesis pathways, including FPS, ZEP, PDS, SDR. Among them, SDR is a differentially expressed gene, and the addition of inhibitors can significantly reduce the expression of this gene. Therefore, ABA is mainly synthesized through the carotenoid pathway in Py. yezoensis.

3. The ABA-mediated antioxidant enzymes and their transcriptional level under the high temperature stress and low salinity conditions were studied. The content of ABA and the activity and expression of antioxidant enzymes were determined under different temperature (15℃, 20℃) and salinity (30, 25, 20). Here, the photosynthesis parameters, the contents of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) were determined in different treatment conditions. The results showed that the photosynthesis system might be damaged under the conditions of low salinity of 20 at 20℃ temperature. Although the accumulation of H₂O₂ was not detected, the MDA was significantly up-regulated in the samples with low salinity. At salinity of 20, the SOD and POD activities of the samples were up-regulated when they were stressed with high temperature of 20℃, which indicated that the oxygen radical generation and scavenging system in algal cells were active. However, all antioxidant enzymes and the related genes determined here were down-regulated under the conditions of salinity 20 and 20℃. Therefore, we speculated that in the cultivation and production of Py. yezoensis in the sea area, the disease rot induced by high temperature is likely to be directly related to the decrease of ABA content and the inhibition of antioxidant enzyme system, which may be one of the physiological basis of the high temperature disease rot of Py. yezoensis in the production process.

4. We found the PyMPV17 gene is differentially expressed under different treatment conditions in transcriptome data. Based on the possible mechanism of ABA mediated up regulation of antioxidant enzymes and functional studies under various stress conditions, the key gene PyMPV17 was screened and overexpressed or silenced strains were obtained by gene editing method, and verified at DNA and RNA levels. In the next step, we will study the stress resistance phenotype and related gene expression to verify the function of the target gene and its application value in the cultivation of new strains of Py. yezoensis.

第一章 绪  论     1

1.1   条斑紫菜概述       1

1.1.1 条斑紫菜的分类地位及分布     1

1.1.2 条斑紫菜生活史  1

1.2   条斑紫菜价值       2

1.2.1 条斑紫菜的营养及经济价值     2

1.2.2 条斑紫菜的人工栽培  3

1.2.3       条斑紫菜烂菜现象分析       3

1.3   条斑紫菜抗氧化机制研究进展    4

1.3.1       植物体中活性氧（ROS）的产生与清除    4

1.3.2       抗氧化系统有效清除逆境中细胞产生的ROS       6

1.3.3       藻类抗氧化机制研究进展    7

1.4   抗逆激素脱落酸（ABA）的研究进展 8

1.4.1       ABA对抗氧化酶表达的调控作用       8

1.4.2       ABA在藻类抗氧化中发挥作用   9

1.4.3       ABA代谢通路解析       10

1.4.4       藻类ABA的合成途径及生理作用      13

1.4.5       MPV17与ROS清除间的关系     13

1.5   研究内容与技术路线    14

1.5.1       研究内容       14

1.5.2       技术路线       15

第二章 ABA在条斑紫菜抗氧化酶活化中发挥重要作用    17

2.1   前言       17

2.2   材料与方法    17

2.2.1       实验材料及处理    17

2.2.2       光合参数的测定    18

2.2.3       ABA含量的测定   19

2.2.4       H2O2含量的测定 19

2.2.5       相关抗氧化酶活性的测定    19

2.3   材料与方法    22

2.3.1       不同处理条件下条斑紫菜光合活性的变化       22

2.3.2       高盐胁迫下不同抑制剂处理条斑紫菜ABA含量的变化       24

2.3.3       不同处理下条斑紫菜H2O2含量的变化    25

2.3.4       不同处理下条斑紫菜抗氧化酶活性的变化       26

2.4   讨论       28

2.4.1       ABA参与条斑紫菜逆境胁迫应激响应       28

2.4.2       ABA对高盐胁迫条件下条斑紫菜光合活性的影响       29

2.4.3       ABA对条斑紫菜H2O2含量的影响   30

2.4.4       ABA在高盐胁迫下调控了相关抗氧化酶活性       31

第三章 条斑紫菜中ABA合成及调控抗氧化酶表达机制分析    33

3.1   前言       33

3.2   材料与方法    33

3.2.1       实验材料的收集    33

3.2.2       文库构建与测序    33

3.2.3       测序数据的质控过滤    34

3.2.4       转录本序列组装与功能注释       34

3.2.5       差异表达基因分析       35

3.2.6       qRT-PCR转录组数据的验证       35

3.3   结果       37

3.3.1       高通量测序数据分析    37

3.3.2       功能基因注释及分类    38

3.3.3       不同样本间差异表达基因数目及KEGG代谢通路注释       39

3.3.4       ABA合成途径中相关基因qRT-PCR结果分析    44

3.3.5       不同抗氧化酶相关基因的qRT-PCR结果分析    45

3.3.6       PyMPV17基因qRT-PCR结果变化     47

3.4   讨论       47

3.4.1       条斑紫菜ABA合成途径分析      47

3.4.2       条斑紫菜ABA合成可能调控了抗氧化酶相关基因的表达    48

3.4.3       PyMPV17基因在条斑紫菜抗氧化中的作用分析       49

第四章 低盐、高温诱导的条斑紫菜病烂发生与ABA及抗氧化酶表达抑制相关       51

4.1   前言       51

4.2   材料与方法    52

4.2.1       实验材料及处理    52

4.2.2       不同处理条件下条斑紫菜光和参数的测定       52

4.2.3       不同处理条件下条斑紫菜体内ABA含量的测定    52

4.2.4       不同处理条件下条斑紫菜H2O2含量的测定       52

4.2.5       丙二醛（MDA）含量的测定      52

4.2.6       不同处理条件下条斑紫菜相关抗氧化酶活性测定       52

4.2.7       抗氧化酶系统关键分子实时荧光定量（qRT-PCR）检测     53

4.2.8       数据分析       54

4.3   结果       54

4.3.1       不同胁迫处理条件下光合参数的变化       54

4.3.2       不同胁迫处理条件下条斑紫菜体内ABA含量的变化    54

4.3.3       过氧化氢及丙二醛含量变化       55

4.3.4       抗氧化酶活性测定结果       56

4.3.5       抗氧化酶系统关键基因的实时荧光定量分析       57

4.3.6       PyMPV17基因实时荧光定量分析      59

4.4   讨论       60

4.4.1       高温、低盐胁迫对条斑紫菜光合作用的影响       60

4.4.2       高温、低盐对条斑紫菜体内ABA含量的影响       60

4.4.3       抗氧化酶活化是条斑紫菜抵御高温胁迫的重要机制之一    61

4.4.4 海水盐度与高温诱发的条斑紫菜病烂发生密切相关       63

第五章 PyMPV17转基因条斑紫菜的分子育种研究 65

5.1   前言       65

5.2   实验材料与方法    66

5.2.1       条斑紫菜叶状体培养    66

5.2.2       RNA提取及反转录制备cDNA    66

5.2.3       PyMPV17基因CDS(coding region)的克隆       66

5.2.4  PyMPV17基因序列及系统发育分析      67

5.2.5       质粒载体构建       68

5.2.6       微粒子弹的构建    69

5.2.7       基因枪转化及藻体轰击后的培养       69

5.3   结果       70

5.3.1       MPV17蛋白序列信息及系统进化发育分析       70

5.3.2       转基因单藻株筛选过程       71

5.3.3       PyMPV17转基因植株DNA水平的验证    72

5.3.4       PyMPV17转基因植株RNA水平的验证    73

5.4   讨论       73

第六章 结论与展望     75

6.1   结论       75

6.2   展望       76

参考文献       77

致  谢    91

作者简历及攻读学位期间发表的学术论文与研究成果       93