实验海洋生物学重点实验室知识库

海带（Saccharina japonica）分布于东亚沿海，是重要的海洋经济褐藻。本研究通过构建高密度遗传连锁图谱，进行了海带长、宽性状QTL定位；对筛选到的候选基因Tic20进行了基因克隆、亚细胞定位、跨膜拓扑结构分析以及蛋白相互作用的研究。相关结果对分析海带长宽性状形成的分子机制，及Tic20在海带中的结构与功能具有重要参考意义。

在SLAF-seq的基础上，选取7627个SNP标记构建了具有31个连锁群，平均遗传距离0.69 cM的遗传连锁图谱；并对海带长、宽性状相关的QTL进行了定位与分析。共检测到12个QTL与叶片长度相关，10个QTL与叶片宽度相关。对海带长、宽增加均有增强效应的相关等位基因均来自亲本。将与长、宽性状均相关QTL的序列比较定位到海带基因组（MEHQ00000000）后，鉴定出14个叶绿体内膜易位子Tic20（Translocons at the inner envelope membranes of chloroplasts 20）基因和3个肽酶s8和s53（Peptidase S8 and S53）基因。荧光定量PCR检测4个Tic20基因的表达水平不仅在两个亲本孢子体中存在差异，而且在同一亲本的不同培养时间也存在差异。此外，与海带长、宽密切相关的SNP标记可用于海带选种，并提高选择效率。

Tic20是一种重要的易位子蛋白，在叶绿体蛋白转运过程中起着重要作用。本研究克隆了海带的Tic20-14基因，SjTic20-14蛋白结构分析发现其由N端非蓝藻起源的EF-hand功能域和C端蓝藻起源的Tic20功能域结合而形成非典型结构。以三角褐指藻（Phaeodactylum tricornutum）为转基因验证模型，证实SjTic20-14定位于三角褐指藻叶绿体最内层膜，蛋白末端N_in-C_in朝向，跨膜拓扑结构为“M”型，EF-hand结构域完全伸入叶绿体基质中。此外，SjTic20-14的转入对三角褐指藻的生长有促进作用。但SjTic20-14在海带中的真实定位和拓扑结构，及其在海带中的功能尚需进一步验证。

以光照处理的海带样品提取RNA，构建了高质量海带膜蛋白酵母双杂交文库。测定文库的库容量为1.44×10⁷ cfu/mL，完整性和覆盖度良好；随机扩增重组率为100%，插入片段0.4~2.6 Kb，插入片段丰富度较高。以SjTic20-14为诱饵进行膜蛋白酵母双杂交筛选互作蛋白，筛库转化总克隆数5.84×10⁶ cfu。在挑取的272个互作酵母菌落中获得99个cDNA序列，其中74个功能已知，25个功能未知。功能互作分析发现SjTic20-14广泛参与各种代谢（磷化合物、氮化合物、碳水化合物和DNA代谢等）、细胞组成（生物膜和动力蛋白）、转录调控、蛋白运输和光合作用等生命过程。

对24对互作进行的回转验证均显示相互作用（阳性率100%）；蛋白结合强度定量检测显示其中17个cDNA序列在分别与Tic20-14全长、EF-hand功能域和Tic20功能域互作时，蛋白结合强度在3者间有显著差异，提示SjTic20-14的结构完整性对其功能有一定影响。用BiFC实验在硅藻中验证到Tic20-14全长、EF-hand功能域和Tic20功能域与捕光复合体亚型6（Light harvesting complex 6, LHC6）均存在互作；间接反映出SjTic20-14可能通过与LHC6互作影响海带光合作用，而影响其长、宽性状的形成，进而影响海带生长。

Saccharina japonica is one of the most important economic brown algae which distributed among the coastal areas of east Asia. In this study, the QTL mapping of S. japonica blade length and width traits carried out by constructing high-density linkage map. The identified candidate Tic20 gene’s functional research was carried out in gene cloning, subcellular localization, transmembrane topology analysis and protein interaction. The results are of great significance for analysing the molecular mechanism of S. japonica blade length and width traits’ formation and the structure and function of Tic20 in S. japonica.

On the basis of SLAF-seq, 7627 SNP markers were selected to construct a linkage map with 31 linkage groups and an average genetic distance of 0.69 cM, and QTLs related to blade length and width phenotypes of S. japonica were located and analyzed. In total, 12 QTLs contributing to blade length and 10 to width were detected. Some QTL intervals were detected for both blade length and width. Additive alleles for increasing blade length and width in S. japonica came from both parents. After the QTL interval regions were comparatively mapped to the current reference genome of S. japonica (MEHQ00000000), 14 Tic20 (translocon on the inner envelope membrane of chloroplast) genes and three peptidase genes were identified. The expression levels of four Tic20 genes were different not only in the two parent sporophytes but also at different cultivation times within one parent. Besides, the SNP markers closely associated with blade length and width could be used to improve the selecting efficiency of S. japonica breeding.

Tic20 is an important translocon protein that plays a role in protein transporting into the chloroplast. In this study, we cloned the S. japonica Tic20-14 gene, structural analysis of SjTic20-14 protein revealed a noncanonical structure consisting of an N-terminal non-cyanobacterium-originated EF-hand domain and a C-terminal cyanobacterium-originated Tic20 domain. Using Phaeodactylum tricornutum as the transgenic verification model, indicated the innermost membrane localization of SjTic20-14 in the P. tricornutum chloroplast, N_in-C_in-terminal orientation with “M”-type transmembrane topology and the EF-hand domain entirely extruded into the chloroplast stroma. In addition, the trangenosis of SjTic20-14 promoted the growth of P. tricornutum. However, the actual localization and topology and further functional analysis of SjTic20-14 in S. japonica is needed.

The high quality S. japonica membrane protein yeast two-hybrid libraries was constructed with light treated high quality S. japonica RNA. The library capacity was 1.44×10⁷ cfu/mL with high library integrity and coverage; recombination rate of random amplification was 100% and the length of inserted fragments ranged from 0.4 to 2.6 Kb, exhibiting a high abundant of inserted fragments. Using SjTic20-14 as bait to screen the interaction proteins with SjTic20-14, the total number of clones co-transformed into sieve libraries were 5.84×10⁶ cfu. Total 99 cDNA sequences were obtained from 272 selected colonies, among which 74 had known functions while 25 with unknown functions. Functional interaction analysis revealed that SjTic20-14 is widely involved in various life course like metabolic processes (phosphate-containing compound metabolism, nitrogen compound metabolism, carbohydrate metabolism and DNA metabolism, etc.), cellular composition (biofilm and dynein), transcriptional regulation, protein transport and photosynthesis.

All the random chose 24 cDNAs for the rotary validation showed positive interaction (100% positive rate). Quantitative detection of protein interacting strength showed significant differences in 3 types of interactions of the 17 in the 24 chose cDNA sequences of full-length SjTic20-14, EF-hand domain and Tic20 domain, respectively; suggesting that the structural integrity of SjTic20-14 had a certain impact on its function. With BiFC validation indicated the interactions Tic20-14, EF-hand and Tic20 domain with the light harvesting complex subtype 6 (LHC6) respectively in P. tricornutum. This also indirectly reflects that SjTic20-14 may affect the photosynthesis of S. japonica by interacting with LHC6, thereby affecting the formation of the phenotype of S. japonica blade length and width, thus affecting the growth of S. japonica.

第1章  引言... 1

1.1  研究背景... 1

1.1.1  海带生物学特征... 1

1.1.2  海带养殖概况... 1

1.1.3  海带种质资源... 2

1.1.4  海带育种... 3

1.2  海带分子标记与选择... 4

1.2.1  分子育种在海带中的应用... 4

1.2.2  分子标记在海带中的应用... 5

1.2.3  海带QTL定位研究... 6

1.3  Tic20与叶绿体蛋白输入... 7

1.3.1  核编码蛋白输入叶绿体... 7

1.3.3  TOC/TIC介导蛋白转运流程... 8

1.3.4  Tic20研究进展... 9

1.4  蛋白亚细胞定位与跨膜拓扑结构研究... 10

1.4.1  蛋白亚细胞定位... 10

1.4.2  蛋白跨膜拓扑结构... 13

1.4.3  Tic20亚细胞定位与跨膜拓扑结构... 15

1.5  蛋白-蛋白相互作用... 16

1.5.1  蛋白互作预测... 16

1.5.2  蛋白互作实验方法... 17

1.5.3  膜蛋白互作研究... 21

1.6  研究目的及意义... 22

第2章  海带遗传连锁图谱构建及长、宽性状QTL定位... 23

前言... 23

2.1  材料与方法... 24

2.1.1  实验材料... 24

2.1.2  主要仪器与试剂... 24

2.1.3  海带BC₁F₂群体构建... 24

2.1.4  海带DNA提取... 25

2.1.5  SLAF测序文库的准备和测序... 26

2.1.6  根据SLAF测序数据和基因分型挖掘SNP标记... 28

2.1.7  遗传连锁图谱构建及海带长、宽性状QTL定位... 29

2.1.8  候选基因挖掘... 29

2.1.9  RNA提取荧光定量PCR分析... 29

2.2  结果... 31

2.2.1  SNP标记的开发和基因分型... 31

2.2.2  遗传连锁图谱构建... 32

2.2.3  海带长、宽性状相关QTL分析... 36

2.2.4  海带长、宽性状相关候选基因筛选... 40

2.3  讨论... 44

2.3.1  海带QTL定位... 44

2.3.2  长、宽性状相关性... 44

2.3.3  候选基因鉴定... 45

第3章  海带Tic20克隆、亚细胞定位及跨膜拓扑结构研究... 46

前言... 46

3.1  材料与方法... 47

3.1.1  实验材料... 47

3.1.2  主要仪器与试剂... 47

3.1.3  基因克隆... 50

3.1.4  序列分析... 55

3.1.5  转基因质粒制备... 55

3.1.6  质粒转硅藻细胞... 61

3.1.7  转基因硅藻检测... 62

3.2  结果... 64

3.2.1  海带Tic20目的基因选定... 64

3.2.2  海带SjTic20-14克隆及序列分析... 64

3.2.3  各生长时期SjTic20-14表达量... 67

3.2.4  SjTic20-14亚细胞定位及跨膜拓扑结构预测... 67

3.2.5  其它物种蛋白结构与拓扑结构预测... 68

3.2.6  进化分析... 70

3.2.7  SjTic20-14全长转基因... 72

3.2.8  SjTic20-14全长Self-assembly GFP实验... 74

3.2.9  功能域逐步删除SjTic20-14在硅藻定位... 76

3.2.10  流式细胞仪检测荧光信号... 81

3.3  讨论... 83

3.3.1  SjTic20-14蛋白结构... 83

3.3.2  SjTic20-14蛋白亚细胞定位及跨膜拓扑结构... 84

第4章  海带SjTic20-14蛋白互作网络构建及功能分析... 86

前言... 86

4.1  材料与方法... 86

4.1.1  实验材料... 86

4.1.2  主要仪器与试剂... 87

4.1.3  膜蛋白酵母文库构建... 88

4.1.4  SjTic20-14诱饵质粒筛库... 99

4.1.5  BiFC法目的蛋白互作验证... 109

4.2  结果... 112

4.2.1  分裂泛素化酵母文库构建... 112

4.2.2  质粒单转酵母... 114

4.2.3  诱饵质粒自激活检测... 115

4.2.4  抑制剂浓度筛选... 117

4.2.5  文库筛选... 118

4.2.6  蛋白互作验证与结合强度检测... 124

4.2.7  BiFC验证SjTic20-14与LHC6的互作... 127

4.3  讨论... 129

4.3.1  分裂泛素化酵母文库构建... 129

4.3.2  海带膜蛋白Tic20互作网络构建... 129

第5章  结论... 131

参考文献... 132

附  录... 151

致  谢... 185

作者简历及攻读学位期间发表的学术论文与研究成果... 186