CsPPR和CsCPN60-like在茶树白化叶片中的表达分析及互作蛋白验证

doi:10.13560/j.cnki.biotech.bull.1985.2022-0802

生物技术通报 ›› 2023, Vol. 39 ›› Issue (3): 218-231.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0802

CsPPR和CsCPN60-like在茶树白化叶片中的表达分析及互作蛋白验证

王涛¹^,²(), 漆思雨¹^,², 韦朝领³, 王艺清¹^,², 戴浩民¹^,², 周喆¹^,², 曹士先⁴, 曾雯¹^,²(), 孙威江¹^,²()

1.福建农林大学园艺学院，福州 350002
2.海峡两岸特色作物安全生产省部共建协同创新中心，福州 350002
3.安徽农业大学茶与食品科技学院，合肥 230000
4.武夷星茶业有限公司，南平 353000

收稿日期:2022-06-29 出版日期:2023-03-26 发布日期:2023-04-10
通讯作者: 曾雯，女，博士，讲师，研究方向：茶树栽培育种与生物技术；E-mail: wenzeng_t@163.com；
孙威江，男，博士，教授，研究方向：茶树种质资源创新与品质化学；E-mail: swj8103@126.com
作者简介:王涛，男，硕士研究生，研究方向：茶树栽培育种与生物技术；E-mail: wangtwtao0827@163.com
基金资助:
茶树生态经济型品种筛选及配套栽培技术(2019YFD1001601);福建省高校产学合作项目(2019N5007)

Expression Analysis and Interaction Protein Validation of CsPPR and CsCPN60-like in Albino Tea Plant（Camellia sinensis）

WANG Tao¹^,²(), QI Si-yu¹^,², WEI Chao-ling³, WANG Yi-qing¹^,², DAI Hao-min¹^,², ZHOU Zhe¹^,², CAO Shi-xian⁴, ZENG Wen¹^,²(), SUN Wei-jiang¹^,²()

1. College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002
2. Ministerial and Provincial Joint Innovation Centre for Safety Production of Cross-Strait Crops, Fujian Agriculture and Forestry University, Fuzhou 350002
3. College of Tea and Food Science and Technology, Anhui Agriculture University, Hefei 230000
4. Wuyi Star Tea Industrial Company Limited, Nanping 353000

Received:2022-06-29 Published:2023-03-26 Online:2023-04-10

摘要/Abstract

摘要：

本课题组前期通过转录组筛选出2个与‘白鸡冠’茶树（Camellia sinensis）叶片白化相关的基因（CSS0013384和CSS0036305），为探明CSS0013384和CSS0036305在白化茶树中的表达模式与其互作蛋白，以‘白鸡冠’茶树叶片为材料，克隆CSS0013384和CSS0036305 cDNA全长序列，利用生物信息学、酵母单杂交和酵母双杂交，分析其蛋白理化性质、系统进化树、染色体定位、基因结构、蛋白结构、蛋白调控与互作网络和基因表达模式。生物信息学分析结果表明，CSS0013384和CSS0036305分别属于三角状五肽蛋白（pentatricopeptide repeat protein, PPR）和伴侣蛋白（chaperone, CPN60-like）家族，其蛋白质编码区（coding sequence, CDS）长度为1 893 bp和1 752 bp，编码氨基酸个数为631和575，蛋白质质量为71.87 kD和60.79 kD，等电点为8.93和6.21。亚细胞定位预测结果表明，CSS0013384定位于叶绿体，CSS0036305定位于线粒体。通过白鸡冠茶树第二叶的遮阴和恢复光照处理与不同叶色茶树品种的RT-qPCR发现，CSS0013384和CSS0036305在白化芽叶中高表达。CSS0002807属于PIF转录因子家族，酵母单杂交结果表明CSS0002807可以结合CSS0013384启动子。CSS0013384和CSS0036305在白化茶树叶片中可能参与叶绿体和线粒体发育，在叶片白化过程中发挥重要作用，结果可为进一步探究茶树叶片白化机理提供参考。

关键词: 茶树, 白化, 三角状五肽蛋白, 伴侣蛋白, 表达分析, 互作蛋白

Abstract:

Our group screened out two genes（CSS0013384 and CSS0036305）related to the albino leaf color of Camellia sinensis Baijiguan through the transcriptome, in order to explore the expression patterns of CSS0013384 and CSS0036305 in albino tea plants interacting proteins. Using cultivar Baijiguan as material, the full-length cDNA sequences of CSS0013384 and CSS0036305 were cloned. Bioinformatics, yeast one-hybrid and yeast two-hybrid were used to analyze the protein physicochemical properties, the phylogenetic tree, the chromosome location, the gene structure, the protein structure, the protein regulation and interaction network, and gene expression pattern. Bioinformatics analysis showed that CSS0013384 and CSS0036305 belonged to the pentatricopeptide repeat protein（PPR）and chaperone（CPN60-like）families, respectively. The lengths of their protein coding sequence（CDS）was 1 893 bp and 1 752 bp, the number of encoded amino acids was 631 and 575, the protein mass was 71.87 kD and 60.79 kD, the isoelectric points were 8.93 and 6.21, the subcellular localization prediction results indicated that CSS0013384 was localized in chloroplast, and CSS0036305 was localized in mitochondria. The RT-qPCR results of the second leaf of Baijiguan under shading and restored light treatment and tea plant varieties with different leaf colors revealed that CSS0013384 and CSS0036305 were highly expressed in albino leaves. CSS0002807 belonged to the PIF transcription factor family, and the yeast one-hybrid results indicated that CSS0002807 bound to the CSS0013384 promoter. CSS0013384 and CSS0036305 may be involved in the development of chloroplast and mitochondria in albino tea leaves, and play an important role in the process of leaf albino. The results may provide a reference for further research on the mechanism of tea leaf albino.

Key words: Camellia sinensis, albino, pentatricopeptide repeat protein, chaperone, expression analysis, interacting proteins

王涛, 漆思雨, 韦朝领, 王艺清, 戴浩民, 周喆, 曹士先, 曾雯, 孙威江. CsPPR和CsCPN60-like在茶树白化叶片中的表达分析及互作蛋白验证[J]. 生物技术通报, 2023, 39(3): 218-231.

WANG Tao, QI Si-yu, WEI Chao-ling, WANG Yi-qing, DAI Hao-min, ZHOU Zhe, CAO Shi-xian, ZENG Wen, SUN Wei-jiang. Expression Analysis and Interaction Protein Validation of CsPPR and CsCPN60-like in Albino Tea Plant（Camellia sinensis）[J]. Biotechnology Bulletin, 2023, 39(3): 218-231.

图/表 11

表1 试验所需引物

Table 1 Primers required for the experiment

基因名称 Gene name	上游引物 Sense primer（5'-3'）	下游引物 Anti-sense primer（5'-3'）	备注 Note
CSS0013384	ATGGCCATCCCCAGTTCTCCAGACTGGTCTGTG	TTACATGGTGCCAAAGCTCTGTTGCAACCATTTTGC	基因克隆引物
CSS0036305	ATGTACAGAGTTGGTGCAGCCTTCGCTTCATCGAT	TCAGTAACCCATATCATCCAAATTTGGCATTCGAT	基因克隆引物
qCSS0013384	TGGGGAAAACAGAAGGGCTAA	CAACACCACCACCTGGAACAA	荧光定量引物
qCSS0036305	CCAAAATGAAGGAAAGAGGGTG	TCAAAGCGGAGCGATAGAGC	荧光定量引物
β-Actin	GCCATCTTTGATTGGAATGG	GGTGCCACAACCTTGATCTT	RNA验证
pAbAi- CSS0013384pro	AAATGATGAATTGAAAAGCTTAAGAAATTTTTTGTTGAG	ATACAGAGCACATGCCTCGAGAATTTTGCCTTGCATCAT	酵母单杂交
AD-CSS0002807	GCCATGGAGGCCAGTGAATTCATGAAAGGAATCATGAA	CTCGATGGATCTCACTTACTTGAGCTAGAA	酵母单杂交
BK-CSS0003881	ATGGCCATGGAGGCCGAATTCATGGCATCAACTTTGGCT	CCGCTGCAGGTCGACGGATCTTAGTAACCGTATCCTGA	酵母双杂交
BK-CSS0041773	ATGGCCATGGAGGCCGAATTCATGGCATCAACTTTGGCT	CCGCTGCAGGTCGACGGATCTTAGTAACCGTATCCTGA	酵母双杂交
BK-CSS0043610	ATGGCCATGGAGGCCGAATTCATGTACCGCTTCGCAG	CCGCTGCAGGTCGACGGATCTCAAAAATCCATACCACC	酵母双杂交
AD-CSS0036305	GCCATGGAGGCCAGTGAATTCATGTACAGAGTTGGTGC	CAGCTCGAGCTCGATGGATCTCAGTAACCCATATCATCC	酵母双杂交
BK-CSS0036305	ATGGCCATGGAGGCCGAATTCATGTACAGAGTTGGTGCA	CCGCTGCAGGTCGACGGATCTCAGTAACCCATATCATCC	酵母双杂交
AD-CSS0003881	GCCATGGAGGCCAGTGAATTCATGGCATCAACTTTGGCT	CAGCTCGAGCTCGATGGATCTTAGTAACCGTATCCTGA	酵母双杂交
AD-CSS0041773	GCCATGGAGGCCAGTGAATTCATGGCATCAACTTTGGCT	CAGCTCGAGCTCGATGGATCTTAGTAACCGTATCCTGA	酵母双杂交
AD-CSS0043610	GCCATGGAGGCCAGTGAATTCATGTACCGCTTCGCAG	CAGCTCGAGCTCGATGGATCTCAAAAATCCATACCAC	酵母双杂交

图1 基因扩增电泳图及蛋白结构预测 a：茶树CSS0013384和CSS0036305 PCR扩增电泳图（M：marker；1：CSS0036305；2：CSS0013384）；b：茶树CSS0013384蛋白保守结构域预测；c：CSS0013384 motif分布图；d：茶树CSS0036305蛋白保守结构域预测

Fig. 1 Gene amplification electropherogram and protein structure prediction a: Electrophoresis of PCR amplification of CSS0013384 and CSS0036305 in C. sinensis. b: Conserved domain prediction of CSS0013384 in C. sinensis. c: Motif distribution map of CSS0013384. d: Conserved domain prediction of CSS0036305 in C. sinensis

表2 CSS0013384和CSS0036305蛋白的基本理化性质

Table 2 Basic physicochemical properties of CSS0013384 and CSS0036305 protein

基因 Gene	分子量 Mw/kD	等电点 pI	不稳定系数 Instability index	脂肪族氨基酸指数Aliphatic index	平均疏水性 GRAVY	信号肽 Signal peptide	跨膜结构 Transmembrane domain	亚细胞定位预测 Subcellular localization
CSS0013384	71.87	8.93	40.73	85.90	-0.167	无	无	叶绿体
CSS0036305	60.79	6.21	21.85	103.99	0.002	无	无	线粒体

图2 基因染色体定位、基因结构及蛋白三级结构 a：茶树CSS0013384和CSS0036305染色体定位；b：茶树CSS0013384和CSS0036305基因结构；c：茶树CSS0013384蛋白三级结构预测；d：茶树CSS0036305蛋白三级结构预测

Fig. 2 Gene chromosomal location, gene structure and protein tertiary structure a: Chromosomal localization of CSS0013384 and CSS0036305 in tea plant（C. sinensis）. b: Gene structure of CSS0013384 and CSS0036305 in tea plant. c: The tertiary structure prediction of CSS0013384 in tea plant. d: The tertiary structure prediction of CSS0036305 in tea plant

图3 基因进化树 a：茶树CSS0013384与不同植物的PPR的进化树分析；b：茶树CSS0036305与不同植物的CPN60-like的进化树分析

Fig. 3 Genetic tree a: PPR neighbor-joining tree of CSS0036305 in tea pant and other plants. b: CPN60-like neighbor-joining tree of CSS0036305 in tea plant and other plants

表3 Prediction of cis-elements in the upstream region of CSS0013384和CSS0036305 in tea plant

Table 3

类型Type	基因名称Gene name		注释Function
类型Type	CSS0013384	CSS0036305	注释Function
光响应 Light response	A-box（2）、ATCT-motif（1）、Box 4（6）、Box Ⅱ（1）、GA-motif（1）、GATA-motif（1）、G-box（5）、I-box（1）	Box 4（1）、Box Ⅱ（1）、GATA-motif（3）、G-box（2）、GT1-motif（2）、TCCC-motif（1）、TCT-motif（2）、	参与光反应的顺式作用元件cis-acting element involved in light responsiveness
转录因子结合位点 Transcription factor binding site	MRE（1）、MBS（1）、MBSI（1）、Myb-binding site（1）	HD-Zip3（1）、MRE（1）、MBS（2）	转录因子结合位点 Transcription factor binding site
激素响应 Hormone response	AAGAA-motif（1）、ABRE（2）、ABRE2（1）	—	参与脱落酸反应的顺式作用元件cis-acting element involved in the abscisic acid responsiveness
	ERE（3）	—	参与乙烯反应的顺式作用元件 cis-acting element involved in ethylene responsiveness
	TCA-element（1）	TCA-element（2）	与水杨酸反应有关的顺式作用元件 cis-acting element involved in salicylic acid responsiveness
	—	as-1（3）、CGTCA-motif（3）、TGACG-motif（3）	参与MeJA反应的顺式作用元件 cis-acting element involved in MeJA responsiveness
组织特异性 Tissue specificity	CAT-box（2）、CCGTCC motif（4）、GCN4 motif（1）	—	组织特异性元件 Tissue specific element
胁迫响应 Stress response	ARE（1）、STRE（1）、WUN-motif（4）	ACE（1）、ARE（4）、STRE（2）、WRE3（2）、WUN-motif（2）	胁迫响应元件Stress response element

图4 茶树不同部位基因的表达模式 a：CSS0013384在茶树不同组织部位表达模式；b：CSS0036305在茶树不同组织部位表达模式。不同小写字母表示在P<0.05水平差异显著

Fig. 4 Expression patterns of genes in different parts of tea plant a: Expression pattern of CSS0013384 in different tissues of tea plant. b: Expression pattern of CSS0036305 in different tissues of tea plant. Different lower letters mean the significant difference in P<0.05

图5 遮阴和恢复光照与不同茶树品种间基因表达模式 a：茶树CSS0013384在遮阴和恢复光照下的表达模式分析；b：茶树CSS0036305在遮阴和恢复光照下的表达模式分析；c：茶树CSS0013384在不同茶树品种中的表达分析；d：茶树CSS0036305在不同茶树品种中的表达分析。N0：不遮阴；S1：遮阴1 d；S3：遮阴3 d；RL1：恢复光照1 d；RL2：恢复光照2 d；MD：‘金牡丹’；AJBC：‘安吉白茶’；BJG：‘白鸡冠’；JG：‘金光’；QNX：‘千年雪’；ZH1：‘中黄1号’；HYZ：‘黄芽早’；HJC：‘黄金茶’；HH：黄化品系；HJY：‘黄金芽’；YJX：‘御金香’。不同字母表示样本间存在显著差异（P<0.05）；遮阴和恢复光照处理下基因的相对表达量以N0为对照，不同茶树品种间的基因表达模式以‘金牡丹’（MD）为对照

Fig. 5 Gene expression patterns of genes from different tea cultivars under shading and restored light a: Expression pattern analysis of tea plant CSS0013384 under shading and restored light. b: Expression pattern analysis of tea plant CSS0036305 under shading and restored light. c: Expression analysis of CSS0013384 in different tea plant varieties. d: Expression analysis of CSS0036305 in different tea plant varieties. N0: No shading. S1: Shading 1 d. S3: Shading 3 d. RL1: Restored light for 1 d. RL2: Restored light for 2 d. MD: Jinmudan. AJBC: Anjibaiha. BJG: Baijiguan. JG: Jinguang. QNX: Qiannianxue. ZH1: Zhonghuang 1. HYZ: Huangyazao. HJC: Huangjincha. HH: Albino tea plant germplasm. HJY: Huangjinya. YJX: Yujinxiang. Different letters indicate significant differences between samples（P<0.05）. Relative expressions of genes under shading and restored light were compared with N0, and the gene expression patterns among different tea varieties were compared with Jinmudan（MD）

图6 基因互作网络预测图 a：茶树CSS0013384蛋白互作网络；b：茶树CSS0036305蛋白互作网络。菱形代表转录因子，圆形代表功能基因

Fig. 6 Gene interaction network prediction map a: Protein interaction network of CSS0013384 in tea plant. b: Protein interaction network of CSS0036305 in tea plant. Diamond represents transcription factor. Circle represents functional gene

表4 CSS0013384和CSS0036305互作蛋白功能注释

Table 4 Functional annotations of interacting proteins of CSS0013384 and CSS0036305

靶蛋白 Target protein	互作蛋白 Interacting protein	基因注释 Gene annotation
CSS0036305	CSS0029920	TCP-1/cpn60 伴侣蛋白家族蛋白 TCP-1/cpn60 chaperonin family protein
	CSS0043610	TCP-1/cpn60 伴侣蛋白家族蛋白 TCP-1/cpn60 chaperonin family protein
	CSS0003881	β Rubisco大亚基结合蛋白亚基 Rubisco large subunit-binding protein subunit beta
	CSS0041773	β Rubisco大亚基结合蛋白亚基 Rubisco large subunit-binding protein subunit beta
	CSS0036200	Rubisco亚基结合蛋白β亚基 Rubisco subunit binding-protein beta subunit
	CSS0044373	GAGA结合转录激活因子 GAGA-binding transcriptional activator
	CSS0007067	GAGA结合转录激活因子 GAGA-binding transcriptional activator
	CSS0008658	GAGA结合转录激活因子 GAGA-binding transcriptional activator
	CSS0046266	MYB_related蛋白 MYB_related protein
	CSS0035646	MYB_related蛋白 MYB_related protein
	CSS0028905	MADS-box蛋白 MADS-box protein
CSS0013384	CSS0002939	鸟嘌呤核苷酸结合蛋白亚基β-蛋白异构体X2 Guanine nucleotide-binding protein subunit beta-like protein isoform X2
	CSS0002807	PIF转录因子 Transcription factor PIF7
	CSS0028905	MADS家族蛋白 MADS-box protein

图7 酵母单杂交和酵母双杂交结果图 a：CSS0013384启动子自激活检测；b：CSS0013384酵母单杂交结果；c：CSS0036305酵母双杂交结果。SD/-Ura+AbA：添加金担子素A的缺尿嘧啶的SD培养基；SD/-Leu+AbA：添加金担子素A的缺亮氨酸SD培养基；SD/-Trp/-Leu：缺色氨酸和亮氨酸SD培养基；SD/-Trp/-Leu/-Ade/-Leu：缺色氨酸、组氨酸、腺嘌呤和亮氨酸SD培养基；SD/-Trp/-Leu/-Ade/-Leu/X-α-gal：添加X-α-Gal的缺色氨酸、组氨酸、腺嘌呤和亮氨酸SD培养基

Fig. 7 Yeast one-hybrid and yeast two-hybrid results a: CSS0013384 promoter self-activation detection. b: Results of yeast one-hybrid for CSS0013384. c: Results of yeast two-hybrid for CSS0036305. SD/-Leu+AbA: SD medium lacking leucine supplemented with aureobasidin A. SD/-Trp/-Leu: SD medium lacking tryptophan and leucine. SD/-Trp/-Leu/- Ade/-Leu: SD medium lacking tryptophan, histidine, adenine and leucine. SD/-Trp/-Leu/-Ade/-Leu/X-α-gal: SD medium lacking tryptophan, histidine, adenine and leucine supplemented with X-α-Gal

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CsPPR和CsCPN60-like在茶树白化叶片中的表达分析及互作蛋白验证

Expression Analysis and Interaction Protein Validation of CsPPR and CsCPN60-like in Albino Tea Plant（Camellia sinensis）

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