Cloning and physicochemical characterization of <i>MePPD3</i> gene from cassava (<i>Manihot esculenta</i>)

JIA Suhang; ZHU Shousong; FU Renwen; LI Chunxia; CHEN Yinhua

doi:10.15886/j.cnki.rdswxb.20230035

Volume 15 Issue 1

Jan. 2024

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JIA Suhang, ZHU Shousong, FU Renwen, LI Chunxia, CHEN Yinhua. Cloning and physicochemical characterization of MePPD3 gene from cassava (Manihot esculenta)[J]. Journal of Tropical Biology, 2024, 15(1): 10-18. doi: 10.15886/j.cnki.rdswxb.20230035

Citation:

JIA Suhang, ZHU Shousong, FU Renwen, LI Chunxia, CHEN Yinhua. Cloning and physicochemical characterization of MePPD3 gene from cassava (Manihot esculenta)[J]. Journal of Tropical Biology, 2024, 15(1): 10-18. doi: 10.15886/j.cnki.rdswxb.20230035

Cloning and physicochemical characterization of MePPD3 gene from cassava (Manihot esculenta)

doi: 10.15886/j.cnki.rdswxb.20230035

College of Tropical Crops, Hainan University, Haikou 570228, China

Received Date: 2023-03-09
Accepted Date: 2023-05-04
Rev Recd Date: 2023-04-23

Available Online: 2023-05-08

Publish Date: 2024-01-16

Abstract

The subcellular localization and expression analysis of Psbp protein MePPD3 (Manihot esculenta PsbP domain-containing protein 3, Phytozome database number: Manes.05G127800) were performed to explore whether MePPD3 is involved in disease resistance of cassava. MePPD3 gene was amplified by RT-PCR. Sequence analysis showed that MePPD3 gene was 807bp in length, encoding 268 amino acids, with PsbP domain located at position 106-266 aa. Bioinformatics analysis of MePPD3 protein was conducted by NetPhos 3.1 Server, SignalP 5.0 Server, TMHMM Servervr, PSIPRED and PHYRE2 online, respectively. The results indicated that MePPD3 protein contained 32 phosphorylation sites, 5 glycosylation sites, and 1 transmembrane domain. The secondary structure of the protein was composed of 21.3% Helix (Helix), 26.5% fold (strand) and 52.2% random curl (loop). The multiple sequence alignment, phylogenetic tree analysis and conserved domain analysis indicated that PPD3 protein had a high genetic relationship among different plants. Subcellular localization showed that MePPD3 protein was localized in chloroplast. QRT-PCR results revealed that the expression level of MePPD3 gene in different cassava tissues was significantly different, and was the highest in mature leaves. In addition, the expression of this gene was induced by Xanthomonas phaseoli pv. manihotis (Xpm), indicating MePPD3 is involved in the resistance of cassava to Xpm. After MePPD3 gene was silenced by VIGS technique, the leaf lesion area of silent plants was significantly smaller than that of the control plants, which implies that MePPD3 negatively regulates cassava resistance to bacterial fusarium wilt caused by Xpm.
- cassava,
- MePPD3,
- bioinformatics analysis,
- Xpm CHN11

References

[1]	黄洁, 李开绵, 叶剑秋, 等. 我国的木薯优势区域概述[J]. 广西农业科学, 2008(1): 104 − 108.
[2]	李开绵, 林雄, 黄洁. 国内外木薯科研发展概况[J]. 热带农业科学, 2001(1): 56 − 60. doi: 10.3969/j.issn.1009-2196.2001.01.011
[3]	FERRARO V, PICCIRILLO C, TOMLINS K, et al. Cassava (Manihot esculenta Crantz) and yam (Dioscorea spp.) crops and their derived foodstuffs: safety, security and nutritional value[J]. Critical Reviews in Food Science and Nutrition, 2016, 56(16): 2714 − 2727. doi: 10.1080/10408398.2014.922045
[4]	PINTO-ZEVALLOS D M, PAREJA M, AMBROGI B G. Current knowledge and future research perspectives on cassava (Manihot esculenta Crantz) chemical defenses: An agroecological view[J]. Phytochemistry, 2016(130): 10 − 21.
[5]	ZHANG M, XIE L, YIN Z, et al. Biorefinery approach for cassava-based industrial wastes: Current status and opportunities[J]. Bioresource Technology, 2016(215): 50 − 62.
[6]	LÓPEZ C E, BERNAL A J. Cassava bacterial blight: Using genomics for the elucidation and management of an old problem[J]. Tropical Plant Biology, 2012, 5(1): 117 − 126.
[7]	ZÁRATE-CHAVES C A, GÓMEZ DE LA CRUZ D, VERDIER V, et al. Cassava diseases caused by Xanthomonas phaseoli pv manihotis and Xanthomonas cassavae[J]. Molecular Plant Pathology, 2021, 22(12): 1520 − 1537.
[8]	BRICKER T M, ROOSE J L, ZHANG P, et al. The PsbP family of proteins[J]. Photosynthesis Research, 2013, 116(2): 235 − 250.
[9]	IFUKU K. The PsbP and PsbQ family proteins in the photosynthetic machinery of chloroplasts[J]. Plant Physiology and Biochemistry, 2014, 81: 108 − 114. doi: 10.1016/j.plaphy.2014.01.001
[10]	BRICKER T M, FRANKEL L K. Auxiliary functions of the PsbO, PsbP and PsbQ proteins of higher plant Photosystem Ⅱ: A critical analysis[J]. Journal of Photochemistry and Photobiology B-biology, 2011, 104(1/2): 165 − 178.
[11]	IFUKU K, ISHIHARA S, SHIMAMOTO R, et al. Structure, function, and evolution of the PsbP protein family in higher plants[J]. Photosynthesis Research, 2008, 98(1/2/3): 427 − 437.
[12]	IFUKU K, NAKATSU T, KATO H, et al. Crystallization and preliminary crystallographic studies on the extrinsic 23 kDa protein in the oxygen-evolving complex of photosystem Ⅱ[J]. Acta Crystallographica Section D, 2003, 59((Pt8)): 1462 − 1463. doi: 10.1107/S0907444903011004
[13]	IDO K, IFUKU K, YAMAMOTO Y, et al. Knockdown of the PsbP protein does not prevent assembly of the dimeric PSⅡ core complex but impairs accumulation of photosystem Ⅱ supercomplexes in tobacco[J]. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2009, 1787(7): 873 − 881. doi: 10.1016/j.bbabio.2009.03.004
[14]	IFUKU K, YAMAMOTO Y, ONO T A, et al. PsbP protein, but not PsbQ protein, is essential for the regulation and stabilization of photosystem Ⅱ in higher plants[J]. Plant Physiology, 2005, 139(3): 1175 − 1184. doi: 10.1104/pp.105.068643
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木薯（Manihot esculenta）是热带地区主要的粮食作物和经济作物，有着“地下粮仓”、“淀粉之王”的称号^[1-2]，木薯在我国主要用于各种工业原料的生产^[3-5]，其市场前景十分巨大。菜豆黄单胞菌木薯萎蔫致病变种（Xanthomonas phaseoli pv. manihotis, Xpm）早已列入我国有害生物名录，它是木薯细菌性枯萎病的病原菌，其能够使木薯叶片褪绿萎蔫甚至全株死亡，可导致木薯减产20%～90%^[6-7]。1991年于类囊体腔中首次发现PsbP蛋白，后经过不断研究发现PsbP蛋白广泛存在于藻类、蓝细菌和高等植物中^[8-9]。目前，已发现的PsbP蛋白分为3大类，分别是PsbP蛋白、类PsbP蛋白PPL（PsbP-like protein）和PsbP结构域蛋白PPD（PsbP-domain protein）^[10-12]。PsbP蛋白是叶绿体光系统Ⅱ（photosystem Ⅱ complex, PSⅡ）的重要组成部分，它在维持钙离子、氯离子浓度方面发挥重要作用；此外，敲除PsbP基因后， PSⅡ的功能将受到一定的影响^[13-15]。科学家在拟南芥（Arabidopsis thaliana）中发现，PPL1能参与 PSⅡ复合体的组装，其和植物适合光照强度变化有关^[16-17]；拟南芥的PPL2缺失突变体中，叶绿体NADH脱氢酶的积累量和活性降低^[17]。Roose等^[8]于2007年从拟南芥基因组中鉴定出7个PPD蛋白，它们分别是PPD1、PPD2、PPD3、PPD4、PPD5、PPD6、PPD7。已有研究发现，PPD1是PSI复合体的组装因子，在拟南芥中，PPD1缺失突变体能导致PSI复合体无法正常组装，从而影响植物的光合作用^[18-19]；在拟南芥中，PPD5蛋白和OST1相互作用并被其磷酸化，使细胞中H₂O₂积累增加并使气孔关闭增强^[20]。但目前尚未有过任何关于PPD3蛋白功能的研究报道。

本研究以‘华南8号’木薯为材料，通过qRT-PCR扩增得到MePPD3（Manihot esculenta psbP domain-containing protein 3）基因，并对该基因进行生物信息学分析；同时，对该基因在木薯不同部位和病原菌Xpm CHN11侵染后不同时间的表达模式进行分析；最后利用TRV介导的基因沉默技术沉默MePPD3基因后测定抗病表型，旨在为进一步研究MePPD3基因的分子机理和木薯抗细菌枯萎病的抗病机理提供基础。

1. 材料与方法

1.1. 菌株

大肠杆菌DH5α感受态细胞和农杆菌GV3101感受态细胞购自上海唯地生物；菜豆黄单胞菌木薯萎蔫致病变种Xanthomonas phaseoli pv. manihotis CHN11由笔者所在实验室提供。

1.2. 植物材料

供试的木薯品种为‘华南8号’（‘SC8’），由笔者所在实验室提供。

1.3. 试剂

多糖多酚植物总RNA提取试剂盒（RNA prep Pure Plant Kit）和RNA反转录试剂盒（FastKing gDNA Dispelling RT SuperMix）购自天根生化科技有限公司；琼脂糖凝胶回收试剂盒和质粒小提试剂盒购自艾德莱。

1.4. 所用引物

从phytozome数据库中下载木薯MePPD3基因的CDS序列，使用Primer Primer 5.0分别设计该基因的全长扩增引物、定量分析引物和亚细胞定位引物。本研究所用引物如表1所示。

引物名称	引物序列（5′−3′）	引物用途
EF1α-F	TGCCATGTTCCGTGGAAAGATG	内参基因引物
EF1α-R	CCCCTAGGTGGAATGTCACAGACAC	内参基因引物
MePPD3-F	CATATGATGGCGTCTGTTTCTTTGCTGTC	扩增目的基因
MePPD3-R	GAATTCTCATGAAATGAACCTGAAGGATG	扩增目的基因
qpcr-MePPD3-F	AGACTGATGAGCTTCGCGTT	定量分析
qpcr-MePPD3-R	TGACTTGAACCCGCCGTTAG	定量分析
1300-MePPD3-F	TTGATACATATGCCCGTCGACATGGCGTCTGTTTCTTTGC	亚细胞定位
1300-MePPD3-R	GCCCTTGCTCACCATGGATCCTGAAATGAACCTGAAGGATG	亚细胞定位

1.5. MePPD3基因cDNA全长序列克隆

取少许木薯叶片于液氮中速冻，用液氮预冷后的研钵进行研磨。使用试剂盒提取木薯RNA并将其反转录为cDNA。以木薯cDNA为PCR模板，以MePPD3-F和MePPD3-R为引物，扩增木薯MePPD3基因序列。待琼脂糖凝胶电泳检测结果无误后，转入植物亚细胞载体pCAMBIA1300-35S-GFP中，挑选阳性单克隆送至楠山生物进行测序。

1.6. MePPD3基因的生物信息学分析

借助SignalP 5.0 Server 对MePPD3蛋白的信号肽进行预测；通过TMHMM Serverv 2.0分析MePPD3蛋白的跨膜结构；利用NetPhos 3.1 Server在线工具（http://www.cbs.dtu.dk/services/NetPhos/）预测MePPD3蛋白的磷酸化位点和糖基化位点；分别使用在线网站PSIPRED.（http://bioinf.cs.ucl.ac.uk/psipred/）和PHYRE2（http://www.sbg.bio.ic.ac.uk/～phyre2/html/page.cgi?id=index）预测MePPD3蛋白的二级结构和三级结构。

通过NCBI序列比对，找到MePPD3蛋白在不同植物中的同源蛋白。通过DNAMAN 8.0软件对这9个同源蛋白进行多序列比对；使用MEGA 11.0软件对这些同源蛋白构建NJ进化树；使用MEME在线网站对MePPD3及其同源蛋白进行保守Motif分析。

1.7. MePPD3基因的亚细胞定位

将构建好的植物亚细胞定位载体pCAMBIA1300-35S-GFP-MePPD3与空载pCAMBIA1300-35S-GFP分别转入GV3101菌株。挑取阳性克隆于28 ℃ 200 r·min⁻¹培养过夜，取适量菌液转移至新的LB（含Kan和Rif）液体培养基中扩大培养。调节OD₆₀₀≈1.0，黑暗静置2.5 h，注射于生长30 d左右的烟草（Nicotiana tabacum）叶片背面，做好标记，培养40 h后于共聚焦显微镜下观察其荧光。

1.8. qRT-PCR检测MePPD3基因在木薯不同部位中的表达水平

使用Primer Primer 5.0设计定量引物qpcr-MePPD3-F/R。分别取长势相同的木薯的顶芽、幼嫩叶片、成熟叶片、叶柄、须根、块根等部位，每个部位至少3个生物学重复，液氮速冻后，使用RNA prep Pure Plant Kit试剂盒提取木薯总RNA，然后再按照FastKing gDNA Dispelling RT SuperMix试剂盒的流程反转录cDNA。

1.9. qRT-PCR检测Xpm CHN11侵染后不同时间MePPD3基因表达水平变化

用OD₆₀₀≈0.05的菜豆黄单胞菌木薯萎蔫致病变种Xpm CHN11侵染多株长势相同的木薯，分别于侵染后0 h、6 h、1 d、3 d、5 d随机剪取叶片，液氮速冻后将其研磨提取RNA，并反转录为cDNA。

1.10. VIGS沉默MePPD3基因后检测木薯抗病性有无变化

构建沉默载体pCsCMV-MePPD3。将其与正对照pCsCMV-B和负对照pCsCMV-A分别转化入GV3101菌株，在LB液体培养基（含Kan和Rif）中培养阳性克隆至OD₆₀₀=1.0。然后注射于木薯‘SC8’叶片背面，24 ℃培养30 d。

在LPGA固体培养基（含Chl）上活化Xpm CHN11菌株，活化2次后，用无菌水清洗菌体并稀释至OD₆₀₀=0.05，用该菌液注射木薯叶片。侵染6 d后，观察叶片表面病斑的扩散情况。

3. 讨　论

叶绿体是植物体所特有的细胞器，它能为植物体的各项生命活动提供能量。PsbP蛋白是叶绿体光系统Ⅱ的重要组成部分，它能参与 PSⅡ的组装^[13-15]。此外，葡萄球菌萄球菌RXLR效应因子RXLR31154通过影响Psbp来降低H₂O₂积累，从而促进葡萄致病^[21]。PPD蛋白是PsbP蛋白家族的一员，目前已发现的PPD蛋白有7种。蛋白在PSI组装过程中发挥重要作用^[18-19]；PPD5蛋白和ROS爆发、气孔关闭有关^[19]。以上研究充分说明了Psbp蛋白在植物光合作用和免疫反应中发挥着一定的作用。但目前关于PPD3蛋白的研究却不是很多。

为了增进对PPD3蛋白的了解，本研究首次从木薯数据库中得到MePPD3基因。MePPD3基因CDS序列全长807 bp，编码268个氨基酸序列。对MePPD3基因进行了一系列的生物信息学分析，结果发现：该蛋白第50～100位存在跨膜结构，第106到266位为PsbP保守结构域。其三级结构由6个螺旋组成，符合PsbP蛋白家族的结构特征。选取8种植物中的同源蛋白序列进行多序列比对、进化树和保守结构域分析，结果发现，PPD3蛋白在不同植物中具有较高的保守性，木薯MePPD3蛋白和橡胶中PPD3蛋白具有相同的保守结构域，且两者的相似性可达99%。因此，推测PPD3蛋白在不同植物中起到的作用一致或相近。此外，通过烟草瞬时表达，证实MePPD3定位于叶绿体，这与PPD3蛋白的叶绿体/线粒体定位信号相符，说明MePPD3可能在叶绿体中发挥功能。另外，本研究通过qRT-PCR检测，发现MePPD3基因在木薯成熟叶片中表达量最高；黄单胞菌Xpm CHN11侵染木薯叶片后，MePPD3表达量呈现上调趋势，初步推断，MePPD3可能参与了木薯的抗病途径；VIGS沉默MePPD3基因后接种结果显示，MePPD3负调控木薯的抗病途径。结合前人研究推测，MePPD3可能通过影响气孔开闭和ROS爆发来参与木薯抗病，是否正确仍需要后续研究来证实。

Reference (21)

[1]	黄洁, 李开绵, 叶剑秋, 等. 我国的木薯优势区域概述[J]. 广西农业科学, 2008(1): 104 − 108.
[2]	李开绵, 林雄, 黄洁. 国内外木薯科研发展概况[J]. 热带农业科学, 2001(1): 56 − 60.
[3]	FERRARO V, PICCIRILLO C, TOMLINS K, et al. Cassava (Manihot esculenta Crantz) and yam (Dioscorea spp.) crops and their derived foodstuffs: safety, security and nutritional value[J]. Critical Reviews in Food Science and Nutrition, 2016, 56(16): 2714 − 2727.
[4]	PINTO-ZEVALLOS D M, PAREJA M, AMBROGI B G. Current knowledge and future research perspectives on cassava (Manihot esculenta Crantz) chemical defenses: An agroecological view[J]. Phytochemistry, 2016(130): 10 − 21.
[5]	ZHANG M, XIE L, YIN Z, et al. Biorefinery approach for cassava-based industrial wastes: Current status and opportunities[J]. Bioresource Technology, 2016(215): 50 − 62.
[6]	LÓPEZ C E, BERNAL A J. Cassava bacterial blight: Using genomics for the elucidation and management of an old problem[J]. Tropical Plant Biology, 2012, 5(1): 117 − 126.
[7]	ZÁRATE-CHAVES C A, GÓMEZ DE LA CRUZ D, VERDIER V, et al. Cassava diseases caused by Xanthomonas phaseoli pv manihotis and Xanthomonas cassavae[J]. Molecular Plant Pathology, 2021, 22(12): 1520 − 1537.
[8]	BRICKER T M, ROOSE J L, ZHANG P, et al. The PsbP family of proteins[J]. Photosynthesis Research, 2013, 116(2): 235 − 250.
[9]	IFUKU K. The PsbP and PsbQ family proteins in the photosynthetic machinery of chloroplasts[J]. Plant Physiology and Biochemistry, 2014, 81: 108 − 114.
[10]	BRICKER T M, FRANKEL L K. Auxiliary functions of the PsbO, PsbP and PsbQ proteins of higher plant Photosystem Ⅱ: A critical analysis[J]. Journal of Photochemistry and Photobiology B-biology, 2011, 104(1/2): 165 − 178.
[11]	IFUKU K, ISHIHARA S, SHIMAMOTO R, et al. Structure, function, and evolution of the PsbP protein family in higher plants[J]. Photosynthesis Research, 2008, 98(1/2/3): 427 − 437.
[12]	IFUKU K, NAKATSU T, KATO H, et al. Crystallization and preliminary crystallographic studies on the extrinsic 23 kDa protein in the oxygen-evolving complex of photosystem Ⅱ[J]. Acta Crystallographica Section D, 2003, 59((Pt8)): 1462 − 1463.
[13]	IDO K, IFUKU K, YAMAMOTO Y, et al. Knockdown of the PsbP protein does not prevent assembly of the dimeric PSⅡ core complex but impairs accumulation of photosystem Ⅱ supercomplexes in tobacco[J]. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2009, 1787(7): 873 − 881.
[14]	IFUKU K, YAMAMOTO Y, ONO T A, et al. PsbP protein, but not PsbQ protein, is essential for the regulation and stabilization of photosystem Ⅱ in higher plants[J]. Plant Physiology, 2005, 139(3): 1175 − 1184.
[15]	YI X, HARGETT S R, LIU H, et al. The PsbP protein is required for photosystem Ⅱ complex assembly/stability and photoautotrophy in Arabidopsis thaliana[J]. Journal of Biological Chemistry, 2007, 282(34): 24833 − 24841.
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物种名称	学名	基因号
木薯	Manihot esculenta	Manes.05G127800.1
橡胶	Hevea brasiliensis	XP_021686524.1
蓖麻	Ricinus communis	XP_015573931.1
胡杨	Populus euphratica	XP_011041160.1
雷公藤	Tripterygium wilfordii	XP_038714823.1
茶树	Camellia sinensis	XP_028073751.1
芒果	Mangifera indica	XP_044475605.1
麻风树	Jatropha curcas	XP_012073570.1
开心果	Pistacia vera	XP_031270258.1

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