1、2024,44(1):7-20.引文格式：郎雅琴，翟晓巧，曹等.泡桐NLR基因家族分析及其对植原体的响应J.西南林业大学学报（自然科学）JOURNAL OF SOUTHWEST FORESTRY UNIVERSITYJan.20242024年1月No.1Vol.44南西业报大学学林第1期第44卷DOI:10.11929/j.swfu.202208042泡桐NLR基因家族分析及其对植原体的响应郎雅琴！翟晓巧2曹喜兵范国强1（1.河南农业大学泡桐研究所，河南郑州450 0 0 2；2.河南省林业科学研究院，河南郑州450 0 0 8）摘要：为研究PfNLR基因家族在白花泡桐抗丛枝病过程中的作用，运
2、用生物信息学方法全面分析了白花泡桐NLR（Pf NLR）家族成员的组成、理化性质、染色体定位、进化、结构、顺式作用元件和组织表达特异性，结合植原体侵染白花泡桐前后的miRNA和转录组的表达量变化，筛选出与抗丛枝病相关的基因。结果表明：白花泡桐中的199个PfNLRs分属6 个亚家族；91.9%的PfNLRs定位在白花泡桐18 条染色体上，其中6 2.8%呈簇状分布，2 3个PfNLRs发生了10 次染色体片段重复事件和1次染色体串联重复事件；PfNLR含激素类和胁迫类元件；miRNA和转录组的关联分析发现泡桐miRNA与PINLR之间存在156 对靶向关系；大多数PfNLRs在芽和根中表达；1
3、6 个PfNLRs在白花泡桐感染植原体前后的表达水平显著差异。进一步分析发现，pf-miRNA482介导的PfNLR181在白花泡桐抗丛枝病中发挥作用，该结果可为解析白花泡桐NLR蛋白的功能提供理论依据。关键词：白花泡桐；NLR家族；植原体；miRNA；转录组中图分类号：S763文献标志码：A文章编号：2 0 95-1914(2 0 2 4)0 1-0 0 0 7-14Analysis of Paulownia NLR Gene Family andtheir Responses to PhytoplasmasLang Yaqin,Zhai Xiaoqiao,Cao Xibing,Fan Gu
4、oqiang(1.Institute of Paulownia,Henan Agricultural University,Zhengzhou Henan 450002,China;2.Henan Academy of Forestry,Zhengzhou Henan 450008,China)Abstract:In order to study the role of PfNLR gene family in Paulownia fortunei resistance to Paulowniawitches broom(PaWB),bioinformatics methods were us
5、ed to comprehensively analyze the composition,physico-chemical properties,chromosomal localization,evolution,structure,cis-acting elements and tissue expression spe-cificity of P.fortunei NLR(PfNLR)family members.Combined with the miRNA and transcriptome data beforeand after phytoplasma infecting Pa
6、ulownia,the genes associated with resistance to PaWB were screened.The res-ults showed that 199 PfNLRs in P.fortunei belonged to 6 subfamilies;91.9%of PfNLRs were located on 18 chro-mosomes of P.fortunei,of which 62.8%were clustered,among them,there were 10 chromosome segment re-peats and 1 chromoso
7、me tandem repeats in 23 PfNLRs;PfNLR contains hormonal and stress elements;integratedtranscriptome and miRNA analysis found that there were 156 pairs of targeted relationships between PfNLRs andmiRNAs;most of PfNLRs were expressed in buds and roots;of which,16 PfNLRs were significantly different be-
8、fore and after phytoplasma infection.Further analysis revealed that PfNLR181 mediated by pf-miRNA482 played收稿日期：2 0 2 2-0 8-2 0；修回日期：2 0 2 2-11-0 8基金项目：中原学者科学家工作室建设专项（豫科2 0 18 18 5）资助。第1作者：郎雅琴（1998），女，硕士研究生。研究方向：泡桐生物技术。Email:。通信作者：范国强（196 4一），男，博士，教授。研究方向：泡桐生物学。Email:。8第44卷西南林业大学学报a role in resistan
9、ce of PaWB.These results could provide a theoretical basis for analyzing the function of PfNLRprotein.Key words:Paulownia fortunei;NLR family;phytoplasma;miRNA;transcriptomeNLR蛋白是细胞内的一类免疫受体，可以直接或间接识别病原物分泌的效应蛋白，激活下游信号通路，引起植物的超敏反应，进而抵御生物胁迫。根据N端结构域不同，将NLR通常分为三大类：Toll白细胞介素受体1蛋白（TIR）-NBS（NBS）-LRR（LRR）、卷曲
10、螺旋蛋白（C C）-NBS-LRR以及缺少N端结构域的NBS-LRR2-3迄今为止，NLR的免疫作用已在多种植物中有了报道4。如沉默棉花中的GhTNL1后再接种大丽轮枝菌（Verticilliumdahliae）会出现严重的萎焉及叶片脱落等黄萎病现象5；过表达GmKP3可以提高大豆对不同病毒的抗性6-7；水稻（O r y z a s a t i v a）CNL蛋白Pb1可以与OsWRKY45相互作用增强水稻对稻瘟菌的抗性8。此外，NLR也可以在miRNA的调控下参与抗病，如大麦（H o r d e u mv u l g a r e）中过表达miRNA9863可以负调控Mlal的表达，削弱大麦对
11、白粉病的抗性9;烟草中过表达nta-MIR6019/6020后，其靶基因N的功能减弱，植物不能产生过敏反应，使植物易感病10；芸苔属植物的bra-miR1885在感染芜菁花叶病毒之后靶向裂解TNL类基因的转录产物，降低植物抵抗力。然而白花泡桐（Pa u l o w n i a f o r t u n e i）NLR（Pf NLR）在白花泡桐抗丛枝病中的作用未见报道，因此深人研究植原体侵染白花泡桐前后PfNLR家族的变化，对揭示白花泡桐抗丛枝病机理具有重要意义。白花泡桐在我国已有2 0 0 0 多年的栽培历史，在防风固沙、园林绿化和改善生态环境中具有重要作用。因其木材不翘不裂、纹理美观，是制作家
12、具、工艺品和乐器的优良材料，大量种植泡桐具有重要的生态和经济价值12。然而在白花泡桐生产中，容易遭受植原体感染患丛枝病，给农林业生产造成了巨大的经济损失13。本课题组前期研究发现植原体侵染白花泡桐前后PfNLR表达量有明显差异，且PJNLR受pfimiRNA的调控14-15,为进一步研究PfNLR在白花泡桐抗丛枝病中的作用，利用白花泡桐基因组数据，鉴定PfNLR家族成员并进行生物信息学分析，结合白花泡桐感染植原体前后的miRNA和转录组数据，筛选与抗丛枝病发生相关的基因，以期为揭示PfNLR在白花泡桐抗丛枝病中的作用提供参考依据。1材料与方法1.1试验材料以河南农业大学林木生物技术实验室培养的
13、白花泡桐健康组培苗（PF）和植原体感染的组培苗（PFI）为试验材料，培养30 d后，剪取泡桐病苗的顶芽与健康苗的根、茎和顶芽保存于-80冰箱中备用16 1.2PfNLR的鉴定和理化性质分析从Pfam数据库（http:/Pfam.sanger.ac.uk）下载NBS的pfam模型（PF00931），将扫描白花泡桐蛋白数据后获得的成员序列构建白花泡桐特有的 NBSpfam模型，再次扫描获得候选成员，E-value 10-20。同时从 TAIR（h t t p s:/w w W.a r a-bidopsis.org/）数据库下载拟南芥的NBS蛋白序列，在白花泡桐的蛋白库进行blast比对获得第2部分
14、候选成员，E-value10-1。2 部分候选成员合并去重复，最终根据结构域确定成员并分类，并按照NLR家族成员在染色体上从小到大的顺序和在染色体上的位置进行命名，未锚定在染色体上的成员最后命名。利用在线软件Prot-Param（h t t p s:/w e b.e x p a s y.o r g/p r o t p a r a m/）分析PfNLR的蛋白理化性质1.3PfNLR的染色体定位及重复事件分析利用TBtools工具中Gene Location Visual-ize绘制PfNLR的染色体分布图，并根据基因簇划定原则确定呈簇状分布的PfNLR17。用TBtools中的MCScanX功能
15、分析获得白花泡桐内以及白花泡桐与拟南芥（Arabidopsis thaliana）之间的基因对18，最后用Circos工具绘制成图。1.4PfNLR的结构和进化分析以及motif预测利用在线工具GSDS2.0（h t t p:/g s d s.c b i.p k t t p s:/me me-s u i t e.o r g/me me/tools/meme）分析获得PfNLR的meme.xml文件，用TBtools对PfNLR的motif和进化树进行可视化分析。使用MAGE7.0.14构建拟南芥和白花泡桐的NLR的NJ（Ne i g h b o r-j o i n i n g）系统发育树，第1
16、期郎雅琴等：泡桐NLR基因家族分析及其对植原体的响应Bootstrap值设为10 0 0，其他参数为系统默认值1.5PfNLR的顺式作用元件分析利用 PlantCare（h t t p:/b i o i n f o r m a t i c s.p s b.u g e n t.be/webtools/plantcare/html/）分析PfNLR启动子上游2000bp的顺式调控元件，并用Excel对顺式作用元件功能以及元件数量进行归类统计和可视化。1.6PfNLR不同部位的表达特异性从各个亚家族中挑选16 个PfNLRs做泡桐健康苗不同部位的实时荧光定量聚合酶链式反应(Real-time qua
17、ntitative polymerase chain reaction,qRT-PCR）分析（表1）；使用植物RNA抽提试剂盒（北京爱普拜生物技术有限公司）提取PF幼苗的顶芽、茎和根的总RNA；PCR反应体系和扩增程序参照Cao等19方法进行。以PfActin为内参基因，2-Ct法计算相对表达量，试验中每个样品3个生物学重复，并用GraphPadPrism分析数据，引物设计选用Primer-BLAST（h t t p s:/www.ncbi.nlm.nih.gov/tools/primer-blast/)。表1PINLR差异基因qRT-PCR所需引物序列Table1Primer sequenc
18、es required for qRT-PCR of differential PfNLR gene基因正向引物(5-3)反向引物（5-3)PfActinAATGGAATCTGCTGGAATACTGAGGACAATGTTACCPfNLR29AGATGGCGAGAATGTGGTGGACGCGGGTTATCTGTGATGGPfNLR33AGGCTGAAAATCTGGCCTCCATTTGCCGTCTCCCATTCGTPJNLR40TGTGCAGATCCTAAAGCAGCAAGGCTTTGCCAGGAGACAAPfNLR62CTGATGTCACCGAGCTTCCATTCCACCCTTTTGGCCTAC
19、CPfNLR71GGACGCATTTCCTTCTGGGAGCAAACAAGCAAGTGCCGTAPfNLR80GGAAGTTGCGGAAGAGGCTAAGCAATTCCCGGTCATCGAAPfNLR111TTTGTGATCCTCCAGATGCCCCCCCAGGAAGGTCTCATTACAPfNLR120ACTCCAAAGAGAGAAAGGATGGGCATCTTCACACATGCACGGPfNLR128TATGCGAAGGGGGTCAGATGATTGCCGAAGGAAGCTCGAAPfNLR149TGCCAAGAAGAGAAGCAGGGGCTTGGGCATGTTGTCAAGGPfNLR158G
20、AAGCAGTTCTCGTGCCTCTGACTTGCGGGTTGATGGAGAPfNLR159CAGCTCTACTTGTGCCGGTTAGTCTTGCGTGTTGTCACCTPfNLR177GCGTCCATGATCTGCTACGAAACGAGGAATACTCAGGCGGPfNLR181TGAGATGGACAGCGGCATACCTGGGAGACACACCAATCCAPfNLR186GTTTCGCTGTTGGGAACCACACCTCAAGTTTGGGCAGCATPfNLR192GTTCTCACAGTTTTACCCGAGGCAGGGAAAGCCAGAGTGGAAG1.7PfNLR对植原体的响应和qR
21、T-PCR验证从NCBI下载白花泡桐感染植原体前后的转录组数据，登录号为SRR11787883、SRR117 8 7 8 94、SRR11787905、SRR117 8 7 9 16、SRR117 8 7 9 2 7 和SRR11787938。根据PfNLR在PF和PFI中的表达，利用TBtools的HeatMap功能绘制FPKM聚类热图14。按照|log2foldchange|2的标准在PJNLR的转录数据中筛选差异基因。提取PF和PFI幼苗顶芽的总RNA，并随机挑选8 个符合差异标准的PfNLRs进行泡桐病健苗的qRT-PCR验证。方法同1.6 的描述。结合本课题组前期的pfmiRNA结果
22、15，利用psRNATarget在线软件（https:/www.zhaolab.org/psRNATarget/analysis?function=2）预测PfNLR 和pf-miRNA的调控关系，错配值为1，选择期望值3，其他分析参数为系统默认。2结果与分析2.1PfNLR的鉴定和理化性质分析白花泡桐中根据是否含有NBS结构域，共鉴定到199个PfNLRs。按照结构域将PfNLR分为6个亚家族：CC-NBS-LRR(CNL)、CC-NBS(CN)、RPW 8-NBS(RN)、T IR-NBS(T N)、NBS-LRR(NL)和NBS(N)，CC类PfNLR占比大于TIR类的PfNLR，其中C
23、N类成员最多（47.7%），同时具有C端、NBS和N端的典型NLR家族成员有41个。然后根据命名顺序依次命名为PfNLR1199。理化性质分析结果表明PfNLR的氨基酸个10第44卷西南林业大学学报数在130 148 1；等电点在4.7 8 9.92，大部分成员是弱酸性；亲水性在-0.46 2 0.137，绝大多数蛋白为亲水蛋白；PfNLR中有17 5个成员的不稳定指数大于40，被归类于不稳定蛋白（表2）。表2 PNLR家族理化性质分析Table2Physical and chemical property analysis of PfNLR family蛋白名称氨基酸等电点不稳定指数平均亲水
24、性蛋白名称氨基酸等电点不稳定指数平均亲水性PfNLR18865.6836.50-0.081PfNLR1014967.9640.16-0.288PfNLR212555.8947.23-0.090PfNLR1028628.8541.360.267PfNLR39506.3346.71-0.223PfNLR1037798.0245.11-0.169PfNLR411136.0050.20-0.063PfNLR1043125.9839.980.393PfNLR510535.3141.030.067PfNLR1058477.5345.73-0.159PfNLR69558.3944.460.172PfNLR1
25、066888.3046.670.209PfNLR79598.2147.10-0.145PfNLR1074365.8237.86-0.210PfNLR812067.5044.31-0.146PfNLR1084798.1650.86-0.144PfNLR913466.5150.41-0.159PfNLR1093109.2040.93-0.180PfNLR1013546.0846.55-0.122PfNLR1108429.0646.86-0.081PfNLR1113875.7152.43-0.135PfNLR1118478.9745.300.217PfNLR129398.6249.37-0.182P
26、fNLR1123968.5739.84-0.261PfNLR137278.8549.94-0.194PfNLR1135139.0443.640.243PfNLR1412046.4051.32-0.091PfNLR1148696.5550.86-0.223PfNLR159108.5649.46-0.136PfNLR1156928.6737.61-0.268PfNLR1611626.0753.54-0.090PfNLR1163865.6644.19-0.441PfNLR178756.3148.18-0.113PfNLR11710026.3344.750.177PfNLR188507.1651.04
27、0.135PfNLR1182519.3636.75-0.366PfNLR197427.8645.74-0.215PfNLR11910735.9747.630.233PfNLR2010486.2143.00-0.084PfNLR12014815.9848.680.203PfNLR2110555.7945.63-0.139PfNLR1214125.4843.060.293PfNLR2210685.5646.85-0.075PfNLR1228526.2851.61-0.144PfNLR234566.3644.39-0.140PfNLR1238516.5150.76-0.181PfNLR2410876
28、.9241.29-0.147PfNLR1243376.0739.13-0.137PfNLR2510866.0743.43-0.126PfNLR1256746.2042.08-0.193PfNLR2611207.1252.65-0.090PfNLR1268597.0953.19-0.181PfNLR2710746.4446.55-0.116PfNLR1279055.3645.890.179PfNLR2810575.4445.85-0.089PfNLR1288485.4547.53-0.158PfNLR298125.5543.50-0.177PfNLR1298747.3144.010.269PfN
29、LR308928.5844.680.263PfNLR1308786.1646.640.186PfNLR318988.1846.68-0.172PfNLR1318506.1844.730.153PfNLR328066.7441.39-0.059PfNLR1328957.4941.320.127PfNLR339175.9245.59-0.326PfNLR1338996.1142.580.187PfNLR349959.1351.08-0.269PfNLR1348728.3451.69-0.190PfNLR358888.7742.99-0.268PfNLR1358366.0854.50-0.051Pf
30、NLR368866.1446.20-0.136PfNLR1366208.2241.08-0.020PfNLR376207.0249.760.027PfNLR1377525.7444.55-0.152PfNLR3810108.8646.82-0.193PfNLR1388617.8745.98-0.135PfNLR398866.6243.12-0.204PfNLR1398746.2947.780.173PfNLR408735.4550.38-0.193PfNLR1408695.7250.84-0.252PfNLR418686.7238.96-0.255PfNLR1418878.3445.030.3
31、03PfNLR428676.1741.210.210PfNLR1428696.0350.89-0.268PfNLR437105.5452.42-0.085PfNLR1437886.6449.58-0.188PfNLR446835.9954.58-0.092PfNLR1447905.4045.36-0.127PfNLR458727.5751.44-0.159PfNLR1458535.7548.23-0.187PfNLR464118.5550.38-0.046PfNLR1468526.5351.56-0.187PfNLR478135.5352.61-0.050PfNLR1478757.0644.2
32、7-0.110PfNLR486676.1045.71-0.169PfNLR1487976.8044.32-0.14611第1期郎雅琴等：泡桐NLR基因家族分析及其对植原体的响应续表2蛋白名称氨基酸等电点不稳定指数平均亲水性蛋白名称氨基酸等电点不稳定指数平均亲水性PfNLR498826.3647.62-0.109PfNLR1496395.8347.91-0.071PfNLR508545.8643.04-0.164PfNLR15010336.6644.56-0.411PfNLR5110436.6646.89-0.113PfNLR15110035.6749.78-0.073PfNLR529297.7
33、742.48-0.264PfNLR1529257.2336.48-0.288PfNLR538986.0846.56-0.140PfNLR1539276.2741.56-0.268PfNLR549236.5547.76-0.220PfNLR15410038.7141.13-0.216PfNLR5510138.1146.03-0.249PfNLR1558218.4239.79-0.019PfNLR569786.4547.17-0.120PfNLR15612785.5439.460.073PfNLR579496.4447.63-0.098PfNLR1575639.2247.160.058PfNLR5
34、82489.7738.370.017PfNLR15814217.2445.36-0.145PfNLR596646.1044.88-0.131PfNLR15913646.5344.09-0.194PfNLR607527.7945.34-0.306PfNLR1607656.1538.04-0.283PfNLR614008.2643.17-0.394PfNLR1619067.7650.06-0.169PfNLR629188.7544.46-0.302PfNLR16210786.0648.26-0.197PfNLR637128.1147.78-0.178PfNLR1639959.2849.12-0.3
35、45PfNLR6410455.6454.34-0.254PfNLR1648877.6147.46-0.169PfNLR6511726.9553.92-0.103PfNLR1651309.9236.71-0.112PfNLR664586.3239.14-0.447PfNLR1668656.1853.94-0.055PfNLR675008.7153.260.462PfNLR16710526.5753.65-0.314PfNLR688759.2441.830.264PfNLR1688776.8450.320.392PfNLR696016.2847.21-0.234PfNLR1699666.5354.
36、30-0.351PfNLR706509.2744.280.129PfNLR1709085.4045.74-0.210PfNLR719639.1246.60-0.292PfNLR1718565.9246.23-0.292PfNLR729068.8648.26-0.287PfNLR1723786.3435.84-0.226PfNLR738018.3643.83-0.122PfNLR1739086.9441.17-0.246PfNLR745937.9346.36-0.115PfNLR1749306.8439.69-0.228PfNLR758816.5747.49-0.209PfNLR17513776
37、.7747.45-0.262PfNLR768548.5748.28-0.215PfNLR1768367.0844.48-0.152PfNLR778126.7740.05-0.098PfNLR1778867.8151.85-0.218PfNLR785609.2343.99-0.351PNLR17812546.4042.72-0.212PfNLR797148.8348.00-0.156PfNLR17912346.2644.70-0.206PfNLR808816.1443.52-0.256PfNLR1809818.5540.75-0.114PfNLR815315.6743.69-0.213PfNLR
38、18112566.1043.36-0.093PfNLR828678.4046.13-0.161PfNLR1828815.8244.04-0.160PfNLR839217.3039.490.322PfNLR1836336.2946.23-0.282PfNLR8410936.4142.69-0.115PfNLR18410935.9645.44-0.330PfNLR856628.9249.17-0.158PfNLR1855175.4934.58-0.173PfNLR8610275.6145.36-0.129PfNLR1868216.5653.91-0.096PfNLR875276.6448.31-0
39、.072PfNLR1874798.3444.89-0.240PfNLR8811135.9645.95-0.164PfNLR1888638.7848.68-0.184PfNLR8910815.7946.20-0.050PfNLR1898098.9836.58-0.103PfNLR905378.7446.31-0.206PfNLR1902795.9330.90-0.376PINLR912674.7832.340.137PfNLR1919318.4450.45-0.067PfNLR924357.6548.31-0.275PfNLR1929296.8254.33-0.180PfNLR938557.58
40、46.88-0.166PfNLR1938777.9937.16-0.240PfNLR947295.8845.10-0.057PfNLR1948636.1346.79-0.129PfNLR9510446.1645.34-0.069PfNLR1959005.6043.47-0.176PfNLR967518.8954.38-0.146PfNLR1966208.2241.08-0.020PfNLR978898.6044.17-0.205PfNLR1977525.7444.55-0.152PfNLR988026.2951.05-0.156PfNLR19810316.9845.86-0.199PfNLR9
41、93345.5738.01-0.192PfNLR1999265.9546.79-0.090PfNLR1008688.4943.77-0.3412.2PfNLR的染色体定位及重复事件分析染色体定位分析显示PFNLR在染色体上的分布有差异（图1）。199个PfNLRs中，有18 3个PfNLRs定位在18 条染色体上（Chr03和Chr12除外），主要分布在Chr08、Ch r 14和Chr19上，CNL和CN类成员主要分布在Chr08、C h r 10 和Chr14上，NL和NBS类基因主要分布在Chr08和Chr14上，RN分布在Chr06上，TNL和TN类基因分布12西南林业大学学报第44卷
42、在 Chr07、Ch r 17 和 Chr18中。18 3个 PfNLRs中有7 7 个PfNLRs组成16 个基因簇分布，38 个PfNLRs组成两两基因之间分布很近的基因小组，共有115个PfNLRs（6 2.8%）存在基因簇或小组中，以Chr14分布的基因簇最多qWChr02Chr03Chr04ChrosChr06Chr07Chro8Chr09Chr10ChroPINLR3SPINLR37*PINLR30PINLR38PINLR40PNLR39PINLR67*PINLRIPINLR6PINLR30PINLR31PNLR40qWSENLR47PINLR61PINLR7PNLR41PINLR
43、8+PINLR32PNLR42PINLR9PINLRILPINLR48PNLR43PINLR63PINLR62*PINLRIOPJNLR12*PINLR18-*PINLR49PNLR449WPNLR4S+INLR6PINLR20LPINLR27FPANLR22PNLR23PINLR5OPINLR68PNLR24PINLR65*PINLR7O.PJNLR33-*PINLRSIPINLR69PNLR25*PJNLR2PINLR75PINLR7IPNLR26PINLR72PINLR27NLRZOPINLR34*PINLRSPINLRS4PONLRTTLPINLR73FPANLR3+PINLR29PI
44、NLR28*PINLRSPINLRS3PINLR66PINLR78PINLR7APINLR4PJNLRSPINLRSO+PINLR13PONLRSOFUPNLRPINLR17PINLR19*PJNLR6OPINLRSSPANLR14PINLRISPNLR16qWSEChr14PINLR94PINLR9OPINLR9SPINLR97PINLR98PNLR1O2PNLR1O3PINLR99PINLRI00FNLRIO4RqWChr1lChr12Chr13PINLRTO1PINLR1OSChr15Chr16Chr17EINLRI38-PNLR160Chr18Chr19Chr20PINLRIO9PIN
45、LRIIOPNLRIIYPINLRISPINLR107PNLRIS2PINLRIS6-PI NLR7 9PINLR8CHPNLR108PINLR112PINLR/53PI NLR157PINLRIS8PINLR161*PINLR167PINLR168+qW*PINLR81PINLR109PNR17PINLRITGPNLRI59PINLR162-PINLR169PINLR170?PINLRI13PINLR163PINLR82PI NLRI 7 YPINLRIZZPINLRIIAPINLRI20*PINLRII3PNLR121PINLR173WPNLRII8hPINLR/27PNLR165-PIN
46、LR164*PINLR174一PI NLR8 3PINLR88+PINLRT19PINLR128P/NLR166P/NLR176+PINLR182PINLR85PINLR8ONLR122PINLR129PINLR178PINLRI75PINLR86PINLROOEFNLRI3PNLRI31PINLR87PLRT30PJNLRI54+PINLR179PINLRI77+PINLR183PINLR84*PINLR93PINLR9INLRI3PLRPNLRI32+PINLRI80PINLR92PINLR181+PINLRI26PNLRT33+PINLR149PANLR134PINLR130PINLRI
47、50*PINLR735PNLRI37PNLRI40PINLR138PINLR/39qW+PNLR147PNLR140PINLR148PNLRI41NBSRPW8-NBSPNLR142NBS-LRRCC-NBSPNLR143PNLR144TIR-NBSCC-NBS-LRRPINLR14S基因簇一基因小组图1PfNLR的染色体定位Fig.1Localization analysis of PfNLR基因复制事件分析表明有2 3个PfNLRs发生基因复制事件，分别分布在Chr06、Ch r 0 8、Ch r 11、Chr13、Ch r 14和Chr19上（图2 a）。其中2 1个PfNLRs为染色体
48、片段重复事件，2 个PNLRs为染色体串联重复事件，且这些基因全部呈簇状分布，说明基因复制事件可能与PfNLR的成簇分布有关。PINLR/40PINLR141PINLR14627.0PINLR13745.0PINLR9924.0PJNLRI40.021.035.0PINLR87PINLR9O18.030.0101001015.025.0101012.020.0Chr1530Chr14Chr169.015.06.0出10.0Chr17Chr133.05.00.0口0.0PINLR84PINLR80PINLR81PINLR167PNLRI8SAT4G23440PINLR79SPINLR1711T4
49、G23440PINLR179AT4G26090Chr201020二山PINLRI10Chr12二10Chro9O0PINLRI4PINLRSOChro7r09PINLR4003PINLR39ChChr08ChroChro60Chr0420Chr07Chros20Chro51Chr06TT1010020102010200020100a.PfNLR的种内共线性分析b.PfNLR与AtNLR的共线性分析图2PINLR的共线性分析Fig.2Collinearity analysis of PfNLRPfNLR与AtNLR的共线性分析表明，共有5对NLR基因存在共线性关系（图2 b）。5对共线基因中，共
50、涉及9个基因，基因之间存在一对多的现象，可能是由于基因之间保守结构域的类13郎雅琴等：泡桐NLR基因家族分析及其对植原体的响应第1期似，如AT4G23440同时与Chr07上的PfNLR34和Chr08上的PfNLR163存在共线性，并且都属于TIR-NBS类基因。说明PfNLR与AtNLR的功能可能比较相似。2.3PfNLR的结构和进化分析以及motif预测PfNLR的基因结构分析结果表明PfNLR的结构复杂多样（图3）。PfNLR的外显子和内含子数目不尽相同，外显子数在12 0 个之间，内含子数在0 19个之间，其中PfNLR9、Pf NLR92、PfNLR94、Pf NLR96、Pf N
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