橘蚜传播率不同的柑橘衰退病毒分离株CPm的表达特性和分子特征

doi:10.16420/j.issn.0513-353x.2020-1004

摘要/Abstract

摘要：

柑橘衰退病毒(citrus tristeza virus,CTV)通过媒介昆虫传播必需要有病毒小外壳蛋白(minor coat protein,CPm)的参与。为解析褐色橘蚜传播率不同的CTV分离株CPm的分子特征和表达特性,通过克隆、测序获得其CPm基因序列,将对应的氨基酸序列运用DNAStar软件进行相似性和序列比对分析,并应用Western blot技术分析了3个传播率不同的中国CTV分离株CPm的表达特性。结果表明,低传播率的分离株CTLJ与其他分离株CPm氨基酸序列差异较大,同源性仅为93.3% ~ 94.2%,氨基酸序列中有14个位点发生变异,其中9个位点是该分离株特有的。尤其有些位点的氨基酸极性和电荷发生了变化,引起了蛋白质二级结构的改变。然而,传播率不同的CTV分离株CPm的表达水平却无显著差异。这些结果揭示了CTV被褐色橘蚜传播的效率与其CPm的表达特性无关,可能与其氨基端序列变异有关。

关键词: 柑橘衰退病毒, 小外壳蛋白, 表达特性, 分子特征

Abstract:

The minor coat protein(CPm)of citrus tristeza virus(CTV)is necessary in virus transmission by the brown citrus aphid[Toxoptera citricida(Kirkaldy)]. The expression levels of CPm in three isolates with high,middle and low aphid transmissibility were conducted by Western blot technique. Then,the CPm sequences of CTLJ and CT16-2 isolates were acquired by RT-PCR,cloning and sequencing. Finally,these amino acid sequences were used to analyze the multiple alignments by the DNAStar program. The result showed that the identity of amino acid sequence of CPm for CTLJ with low aphid transmissibility was only 93.3%-94.2%,when compared with those of other CTV isolates. Comparison of the CPm of these isolates showed that nine of fourteen amino acid mutation site were unique in the CTLJ isolate. Particularly,some mutations led to substitution of new amino acids with a different charge and polarity,which resulted in changes of the secondary structure of the CPm protein in CTLJ. However,the expression of CPm for CTLJ,CT16-2 and CT11A did not show a significant difference. The results suggest that the aphid transmission efficiency of CTV might be not related to the expression level of CPm,but might be associated with amino acid site variation.

Key words: citrus tristeza virus(CTV), minor coat protein(CPm), expression level, molecular characteristics

中图分类号:

S666.2

刘金香, 王欢欢, 王洪苏, 周彦, 李中安, 周常勇. 橘蚜传播率不同的柑橘衰退病毒分离株CPm的表达特性和分子特征[J]. 园艺学报, 2021, 48(5): 1023-1030.

LIU Jinxiang, WANG Huanhuan, WANG Hongsu, ZHOU Yan, LI Zhongan, ZHOU Changyong. Molecular Characteristic and Expression Level of Minor Coat Protein of Citrus Tristeza Virus Isolates with Different Transmissibility by Toxoptera citricida[J]. Acta Horticulturae Sinica, 2021, 48(5): 1023-1030.

图/表 5

表1 本试验中所用的和NCBI中已确定褐色橘蚜传播率的CTV分离株

Table 1 The CTV isolate from China used in this study and other CTV strains available in GenBank

来源 Origin	CTV分离株 Isolate	NCBI登录号 Accession No.	传播率/% Transmission
美国USA(Harper et al.,2016)	T36	AY170468.1	0.5
美国USA(Harper et al.,2018)	T30	AF260651.1	2.0
美国USA(Harper et al.,2018)	FL202-VT	KC517493.1	31.1
美国USA(Harper et al.,2018)	T68-1	JQ965169.1	50.1
中国 China(Liu et al.,2019)	CTLJ	MW533120	4.4
中国 China(Liu et al.,2019)	CT16-2	MW533121	15.6
中国 China(Liu et al.,2019)	CT11A	JQ911664.1	65.6

图1 甜橙嫩皮中3个CTV分离株CT11A、CT16-2和CTLJ的CPm的表达对照:健康甜橙嫩皮作为对照。相同字母表示差异不显著(P > 0.05)。

Fig. 1 The expression level of CPm of three CTV isolates for CTLJ,CT16-2 and CT11A in the tender bark of sweet orange Control:Healthy sweet orange as control. The same letter indicates no significant different(P > 0.05).

图2 CTV的7个分离株CPm氨基酸序列系统进化树标尺下方的数字表示序列变异度;分支上的数字表示自展支持率。

Fig. 2 Phylogenetic tree of the amino acid sequences of CPm of the seven CTV isolates The number under the distance scale representa sequence viriation degree. The number near branches indicate bootstrap values.

图3 CTV的7个分离株CPm氨基酸序列比对分析相同位点一致的氨基酸没有显示,用“·”代替。

Fig. 3 Multiple sequence alignment of amino acid sequences of CPm of the seven CTV isolates Hide residues that match the consensus exactly as“·”.

图4 CTLJ(低传播率)和CT11A(高传播率)的CPm二级结构比较 H:α-螺旋;E:β-折叠;L:环状结构;AA:氨基酸;OBS sec:试验证明了的二级结构;PROF sec:PROF预测的二级结构;Rel sec:PROF预测可信度;SUB sec:PROF预测的子集。

Fig. 4 Comparison of predicted secondary structure of CPm of CTLJ(the lowly transmissible)and CT11A(the highly transmissible)isolates H:α-Helix;E:β-Extend(sheet);L:Loop;AA:Amino acid;OBS sec:Observed secondary structure;PROF sec:PROF predicted secondary structure;Rel sec:Reliability index for PROF sec prediction;SUB sec:Subset of the PROF sec prediction.

参考文献 31

[1]	Atreya P L, Atreya C D, Pirone T D. 1991. Amino acids substitutions in the coat protein result in loss of insect transmissibility of a plant virus. Proceedings of the National Academy of Sciences of the United States of America, 88:7887-7891.
[2]	Atreya P L, Lopez-Moya J J, Chu M, Atreya C D, Pirone T P. 1995. Mutational analysis of the coat protein N-terminal amino acids involved in Potyvirus transmission by aphid. Journal of General Virology, 76(2):265-270. doi: 10.1099/0022-1317-76-2-265 URL
[3]	Bar-Joseph M, Marcus R, Lee R F. 1989. The continuous challenge of citrus tristeza virus control. Annual Review Phytopathology, 27:291-316. doi: 10.1146/annurev.py.27.090189.001451 URL
[4]	Barzegar A, Rahimian H, Sohi H H. 2010. Comparison of the minor coat protein gene sequences of aphid-transmissible and-nontransmissible isolates of citrus tristeza virus. Journal of General Plant Pathology, 76(2):143-151. doi: 10.1007/s10327-009-0216-7 URL
[5]	Callaway A, Giesman-Cookmeyer D, Gillock E T, Sit T L, Lommel S A. 2001. The multifunctional capsid proteins of plant RNA viruses. Annual Review of Phytopathology, 39:419-460. pmid: 11701872
[6]	Chen A, Walker G, Carter D, Ng J. 2011. A virus capsid component mediates virion retention and transmission by its insect vector. Proceedings of the National Academy of Sciences of the United States of America, 108:16777-16782.
[7]	Damerval C, Vienne D D, Zivy M, Thiellement H. 1986. Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins. Electrophoresis, 7(1):52-54. doi: 10.1002/(ISSN)1522-2683 URL
[8]	Drucker M, Froissart R, Hebrard E, Uzest M, Ravallec M Esperandieu P, Mani J C, Pugnière M, Roquet F, Fereres A, Blanc S. 2002. Intracellular distribution of viral gene products regulates a complex mechanism of cauliflower mosaic virus acquisition by its aphid vector. Proceedings of the National Academy of Sciences of the United States of America, 99:2422-2427.
[9]	Fajinmi A A. 2019. Interactive Effect of blackeye cowpea mosaic virus and cucumber mosaic virus on Vigna unguiculata. Horticultural Plant Journal, 5(2):88-92. doi: 10.1016/j.hpj.2019.01.001 URL
[10]	Harper S J, Cowell S J, Dawson W O. 2018. Bottlenecks and complementation in the aphid transmission of citrus tristeza virus populations. Archives of Virology, 163:3373-3376. doi: 10.1007/s00705-018-4009-1 URL
[11]	Harper S J, Killiny N, Tatineni S, Gowda S, Cowell S J, Shilts T, Dawson W O. 2016. Sequence variation in two genes determines the efficacy of transmission of citrus tristeza virus by the brown citrus aphid. Archives of Virology, 161:3555-3559. doi: 10.1007/s00705-016-3070-x URL
[12]	Karasev A V, Boyko V P, Gowda S, Nikolaeva O V, Hilf M E, Koonin E V, Niblett C L, Cline K, Gumpf D J, Lee R F, Garnsey S M, Lewandowski D J, Dawson W O. 1995. Complete sequence of the citrus tristeza virus RNA genome. Virology, 208:511-520. doi: 10.1006/viro.1995.1182 URL
[13]	Karasev A V, Hilf M E, Garnsey S M, Dawson W O. 1997. Transcriptional strategy of Closteroviruses:mapping the 5′ termini of the citrus tristeza virus subgenomic RNAs. Journal of Virology, 71(8):6233-6236. doi: 10.1128/jvi.71.8.6233-6236.1997 URL
[14]	Killiny N, Harper S J, Alfaress S, Mohtar C E, Dawson W O. 2016. Minor coat and heat-shock proteins are involved in binding of citrus tristeza virus to the foregut of its aphid vector,Toxoptera citricida. Applied and Environmental Microbiology, 82:1914-1916.
[15]	Leh V, Jacquot E, Geldreich A, Haas M, Blanc S, Keller M, Yot P. 2001. Interaction between the open reading frame III product and the coat protein is required for transmission of cauliflower mosaic virus by aphids. Journal of Virology, 75(1):100-106. doi: 10.1128/JVI.75.1.100-106.2001 URL
[16]	Li Ling-di, Zhou Chang-yong, Tian Xiao, Wang Yong-jiang, Tang Ke-zhi, Zhou Yan, Li Zhong-an, Liu Jin-xiang. 2013. Development and application of a real-time RT-PCR approach for quantification of CTV in Toxoptera citricida . Scientia Agricultura Sinica, 46(3):525-533. (in Chinese)
	李玲娣, 周常勇, 田晓, 王永江, 唐科志, 周彦, 李中安, 刘金香. 2013. 褐色橘蚜中CTV实时荧光定量RT-PCR检测方法的建立及应用. 中国农业科学, 46(3):525-533.
[17]	Liu J, Li L, Zhao H, Zhou Y, Wang H, Li Z, Zhou C. 2019. Titer variation of citrus tristeza virus in aphids at different acquisition access periods and its association with transmission efficiency. Plant Disease, 103(5):874-879. doi: 10.1094/PDIS-05-18-0811-RE URL
[18]	Liu S, He X, Park G, Josefsson C, Perry K L. 2002. A conserved capsid protein surface domain of cucumber mosaic virus is essential for efficient aphid vector transmission. Journal of Virology, 76(19):9756-9762. doi: 10.1128/JVI.76.19.9756-9762.2002 URL
[19]	Moreno A, Hébrard E, Uzest M, Blanc S, Fereres A. 2005. A single amino acid position in the helper component of cauliflower mosaic virus can change the spectrum of transmitting vector species. Journal of Virology, 79:13587-13593. doi: 10.1128/JVI.79.21.13587-13593.2005 URL
[20]	Moreno P, Ambrós S, Albiach-Martí M R, Guerri J, Pena L. 2008. Citrus tristeza virus:a pathogen that changed the course of the citrus industry. Molecular Plant Pathology, 9(2):251-268. doi: 10.1111/mpp.2008.9.issue-2 URL
[21]	Ng J C, Falk B W. 2006. Virus-vector interactions mediating nonpersistent and semipersistent transmission of plant viruses. Annual Review of Phytopathology, 44:183-212. doi: 10.1146/annurev.phyto.44.070505.143325 URL
[22]	Roistacher C N, Bar-Jospeh M. 1987. Aphid transmission of citrus tristeza virus:a review. Phytophylactica, 19(2):163-167.
[23]	Ruiz-Ferrer V, Boskovic J, Alfonso C, Rivas G, Llorca O, López-Abella D, López-Moya J J. 2005. Structural analysis of tobacco potyvirus HC-pro oligomers involved in aphid transmission. Journal of Virology, 79(6):3758-3765. doi: 10.1128/JVI.79.6.3758-3765.2005 URL
[24]	Satyanarayana T, Gowda S, Ayllon M A, Dawson W O. 2004. Closterovirus bipolar virion:evidence for initiation of assembly by minor coat protein and its restriction to the genomic RNA 5’ region. Proceedings of the National Academy of Sciences of the United States of America, 101(3):799-804.
[25]	Sharma S R. 1989. Factors affecting vector transmission of citrus tristeza virus in south Africa. Zentralblatt Für Mikrobiologie, 144:283-294. doi: 10.1016/S0232-4393(89)80092-7 URL
[26]	Song Zhen, Zou Min, Xu Xiao-feng, Tang Ke-zhi, Liu Ying, Zhou Chang-yong. 2005. DTBIA of citrus tristeza virus detection in new and old leaves of pomelo. Southwest Horticulture, 33(z1):43-45. (in Chinese).
	宋震, 邹敏, 徐小峰, 唐科志, 刘英, 周常勇. 2005. 柚新老枝叶中柑橘衰退病毒的 DTBIA 检测. 西南园艺, 33(z1):43-45.
[27]	Tian Xiao, Li Ling-di, Liu Jin-xiang. 2013. Research progress of the mechanisms of semipersistent viruses transmission by vectors. Biotechnology Bulletin,(7):48-53. (in Chinese).
	田晓, 李玲娣, 刘金香. 2013. 半持久性病毒的介体传播机制研究进展. 生物技术通报,(7):48-53.
[28]	Woolston C J, Covey S N, Penswick J R, Davies J W. 1983. Aphid transmission and a polypeptide are specified by a defined region of the cauliflower mosaic virus genome. Gene, 23(1):15-23. pmid: 6311674
[29]	Xu Xiao-feng, Zhou Chang-yong, Song Zhen, Wang Xue-feng, Zhou Yan. 2007. Molecular characteristics of CP genes of 4 isolates of citrus tristeza virus and their sub-isolates after single aphid transmission. Acta Phytopathologica Sinica, 37(1):25-30. (in Chinese).
	徐小峰, 周常勇, 宋震, 王雪峰, 周彦. 2007. 4种柑橘衰退病毒源单蚜传毒分离株CP基因的分子特征. 植物病理学报, 37(1):25-30.
[30]	Zhou Y, Zhou C Y, Wang X F, Liu Y Q, Liu K H, Zou Q, Xiang Y, Li Z A. 2011. Influence of the quantity and variability of citrus tristeza virus on transmissibility by single Toxoptera citricida. Journal of Plant Pathology, 93(1):97-103.
[31]	Zhou Yan, Zhou Chang-yong, Wang Xue-feng, Liu Ke-hong, Song Zhen, Li Zhong-an. 2007. Effect of virus sources and host plants on the transmissibility of citrus tristeza virus by single aphid of Toxoptera citricida . Acta Phytophylacica Sinica, 34(6):597-600. (in Chinese)
	周彦, 周常勇, 王雪峰, 刘科宏, 宋震, 李中安. 2007. 毒源和寄主对褐色桔蚜传播柑桔衰退病毒效率的影响. 植物保护学报, 34(6):597-600.