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百合野生原种被分为7个组，百合组内杂交培育出诸多系内杂种品系，例如亚洲百合(Lilium Asiatic hybrids)杂种系、东方百合杂种系(Lilium Oriental hybrids)、麝香百合杂种系(Lilium Longiflorum hybrids)等。进行百合组(系)间杂交育种，将有可能培育出抗逆性更强、观赏性状更加新颖的系间杂种，例如OA杂种系(Lilium Oriental×Asiatic hybrids)、OT杂种系(Lilium Oriental×Trumpet hybrids)[1]。但是，百合组(系)间杂交障碍大，克服组(系)间杂交障碍一直是百合育种需要面对的难题。挖掘百合雌蕊中影响花粉管定向生长的基因，将为阐明百合不亲和机制、克服杂交障碍提供依据。
现代生殖生物学研究认为，雌蕊特异或优势表达基因，影响着花粉管与雌蕊的互作，从而影响着杂交亲和与不亲和性。花粉在柱头表面的粘附、水合、萌发，花粉管生长进入柱头，在柱头和花柱中的生长，进入子房，到达胚珠并释放出两个精细胞，完成双受精。这些步骤需要植物雌雄生殖器官之间快速而有效的信息交流，雌蕊在这种交流中起着更关键的物理和化学桥梁作用。花粉管粘附阶段，已发现芸苔属(Brassica)植物柱头中特异表达的SLR1和SLG蛋白与花粉管包被蛋白相互作用，影响花粉管粘附。花粉管进入柱头和花柱后，某些植物雌蕊中特异或优势表达AGPs、TTS、CRPs、SCA、LTPs、Chemocyanin、Plantacyanin、STIL、S-RNase等多肽或蛋白影响了花粉管粘附、伸长、定向或自交不亲和等生物学过程[2]。花粉管从引导组织沿珠柄到达珠孔附近，由珠孔进入卵器，此两个阶段统称为胚珠导向阶段；在胚珠导向阶段，卵细胞和助细胞特异表达的EA1(Egg Apparatus 1)[3]、助细胞特异表达的LUREs[4-5]被认为参与了花粉管珠孔定向调控。
近年来，人们采用高通量基因挖掘技术及其他分子技术，发现了许多雌蕊中特异或优势表达的基因，可以将其分为以下11个类型：与柱头细胞壁相关，包括类伸展蛋白(extensin-like proteins)[6-7]、果胶酯酶(Pectinesterase)[8-9]；与信号转导相关，包括富含亮氨酸的重复单位(leucine-rich repeat，RLKs)[10-11]、γ-氨基丁酸(GABA)[12-13]、长链脂类[14]；与胁迫和防御相关，包括γ-硫瑾、植物凝集素、胰蛋白酶抑制剂、多酚氧化酶、(1-3)-β-葡糖苷酶[15]、活性氧(ROS)[16-18]；与物质转运相关，包括TTS(transmitting tissue-specific proteins) [19]、ABC转运蛋白[20-21]；与植物激素相关，包括生长素相关基因[22-24]；与控制细胞骨架相关[25-27]；与能量代谢相关，如ADP葡萄糖焦磷酸化酶小亚基、ADP葡萄糖焦磷酸化酶大亚基、UDP葡萄糖焦磷酸化酶[28-29]；与花粉与柱头互作相关，如S-位点类受体激酶(SRK)[30-31]、受体蛋白激酶(receptor-like protein kinases，RLK)[32-37]、泛素连接酶E3[38]；与营养物质相关，如查尔酮合成酶[39]、寡肽/小肽转运和氨基酸转运相关基因[40]；与离子交换相关，如植物细胞内的质子泵相关基因[41-42]；与蛋白命运相关，如泛素(ubiquitin)[43]。这些为阐明植物生殖生物学相关机制奠定了基础。
本研究以东方百合为母本，其雌蕊分别经过亲和性杂交和不亲和性杂交后，采用抑制消减杂交技术(SSH技术)，寻找两组雌蕊中的差异表达基因。以期挖掘雌蕊中与杂交不亲和性相关的基因，为阐明百合组(系)间杂交不亲和机制提供依据。
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试验材料东方百合(Lilium Oriental hybrids)品种‘Sheila’和‘Justina’购自北京山谷园艺公司，野生百合山丹(Lilium pumilum)(卷瓣组)采自北京昌平流村镇老峪沟村。试验材料均种植在北京农学院温室。母本‘Sheila’于开花前1天去雄、套袋；当其柱头出现大量分泌液时，分别取‘Justina’、山丹新鲜花粉进行授粉，其中前者为亲和性杂交，后者为不亲和性杂交。利用荧光显微技术，观察并确定亲和性杂交2 d后，花粉管已穿越母本柱头中的位置；为避免花粉管的干扰，去掉花粉管穿越过的柱头及花柱。以此位置为界线，去掉花粉管穿越过的柱头及花柱，柱头剩余部分及子房作为驱动组(driver)材料。不亲和性杂交2 d后，参考亲和性杂交，去掉相应长度的柱头和花柱，柱头剩余部分及子房作为试验组(tester)材料。
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各收集20个drive雌蕊和20个tester雌蕊，用Plant RNAzol提取总RNA。通过紫外检测OD260/OD280值评估提取的RNA质量，1.0%琼脂糖凝胶电泳鉴定其完整性。用PolyA Tract SystemsⅢ(Z5300)试剂盒从总RNA中分离mRNA。
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以不亲和性杂交后的雌蕊为tester，以亲和性杂交后的雌蕊为driver。参照试剂盒(Clontech PCR-SelectTM cDNA Substraction Kit、SMARTerTM PCR cDNA Synthesis Kit、Advantage 2 PCR kit)说明书构建抑制消减文库。经过cDNA第1链、第2链的合成，RSaⅠ酶切，接头连接，第1轮、第2轮杂交，第1轮、第2轮PCR扩增，正向消减cDNA片段纯化等程序。
取消减cDNA片段纯化4 μL(10 μL反应体系)，连接到pMD-19T载体上，4 ℃过夜。将连接产物转化到100 μL大肠杆菌感受态细胞DH5a(天根)中，冰浴30 min，42 ℃水浴热击60 s，冰浴2 min；从冰上取出，加900 μL LB液体培养基；于37 ℃恒温摇床中，220 r/min，培养1 h；10 000 r/min，室温离心1 min，收集细菌；弃上清900 μL，用剩余液体沉淀重悬，全部涂于Amp/LB培养板，37 ℃，倒置培养16 h，进行蓝白斑筛选。挑取所有白色菌落，加入到含有Amp的LB液体培养基的96孔细胞培养板中，37 ℃，220 r/min，振荡培养过夜。
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以重组子阳性菌菌落的菌液为模板，用pMD19-T载体通用引物(M13-47: 5-CGCCAGGGTTTTCCC AGTCACGAC-3，M13-48: 5-AGCGGATAACAATTT CACACAGGA-3)对菌液进行PCR扩增。反应程序为：94 ℃，3 min；94 ℃，30 s；52 ℃，30 s；72 ℃，1 min，35个循环；72 ℃，10 min；4 ℃冰箱保存。取5 μL PCR产物到1.2%琼脂糖凝胶电泳检测。
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选取差异表达重组子送北京六合华大基因有限公司进行测序，DNAStar分析序列，消除载体及接头序接，拼接，用BLAST软件进行同源性比较，通过Gene Ontology进行功能分类。
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随机挑选10个上述差异基因，根据EST序列设计特异性引物，采用半定量RT-PCR技术，检测其在tester、driver、母本柱头、花柱、子房、花瓣、叶、茎、鳞茎的表达量，其中除tester外，其余材料均取材于亲和性杂交植株。PCR反应条件：94 ℃预变性5 min；94 ℃，变性40 s(见表 1)退火30 s，72 ℃延伸40 s，35个循环；72 ℃延伸10 min。取5 μL PCR产物在1.2%琼脂糖凝胶检测扩增结果。
表 1 特异性引物
Table 1. Specific primer
基因名称
Gene name上游引物(5′-3′)
Forward primer(5′-3′)下游引物(5′-3′)
Reverse primer(5′-3′)退火温度
Annealing
temperature/℃60S核糖体蛋白
60S ribosomal protein(RP)AATCCCTTGAATCCTCTTGCC AGAGAAGGCGAAGATGGTG 54 液泡膜H+-ATPase
Vacuolar H+-ATPase(V-H+-ATPase)AGAGAGAAGATGACCTGAATGAAAT CCAAACGATGCTTGATGACG 54 小G结合蛋白
Small GTP-binding protein (small GTP)ACCTGCTCAAGAACTAGAAG AGTAGGGAACAAAACAAACAC 51 丝氨酸/苏氨酸蛋白磷酸酶
Serine/Threonine-protein phosphatase 2A catalytic subunit3(PP2A-3)GGAGGAAAAGATGAGCGGG AATAATAGCCACGGTCCACATAATC 55 分子伴侣CLPB
Molecular chaperone CLPB(CLPB)TGACTATGCTGTTGATCTGC ATGCAGTTTCGAGATTGAT 49 过氧化氢酶2
Catalase2(CAT2)CAACCTGGAGAGCGATACC GTCGAAGGTAGTAAGCC 50 质膜ATPase
Plasma membrane atpase (PM-ATPase)AGTTTAATGCAAGCGATAT AGCAAGAAGAATTATGGG 48 焦磷酸盐能膜质子泵3
Pyrophosphate-energized membrane
proton pump 3AATCCCTTATTCCACAAACAAG ATCTTCGTTGACCTGGCTAAG 52 肌动蛋白解聚合因子
Actin depolymerizing factor(ADF)GTACCCAAACAAGAAGCACAT TGATGTGTCGAGGGTGAGGAGT 53 钙依赖蛋白激酶Calcium-dependent protein kinase(CDPK) CAGTATTAGAACTCATTGGCAC GTAATCCCCATATTCACTGCTG 54 -
经纯化后的总RNA经过琼脂糖凝胶电泳检测(图 1A)，发现28S:18S值接近2 :1，带型分明，无DNA污染；经过紫外检测，两份材料的OD260/OD280都处于1.8~2.0之间，表明总RNA纯度高、完整性好，可以用于mRNA分离。mRNA电泳检测结果见图 1B，由图可见，纯化后的mRNA呈smear状，完整性较好。
图 1 总RNA和mRNA电泳检测
Figure 1. Electrophoresis detection of total RNA and mRNA
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经质量检测合格的mRNA，先反转录成双链cDNA，再进行RsaⅠ酶切。反转录得到的cDNA经琼脂糖凝胶电泳检测结果如图 2的T1、D1所示，所得cDNA条带呈弥散状。经RsaⅠ酶切过后所得到的cDNA琼脂糖凝胶电泳结果如图 2的T2、D2所示，cDNA条带下移，分子量变小，条带呈弥散状。可见cDNA已经被切开成为利于杂交的片段，可以用于下一步试验。
图 2 cDNA酶切前后的电泳图
Figure 2. Electrophoresis pattern of cDNA before and after digestion
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差异性筛选消减cDNA文库中的消减片段，经两轮PCR扩增后，二次PCR产物电泳结果如图 3所示。pE1中弥散条带亮度明显高于E1，而1-c、p1-c亮度差异较小，说明差异片段得到了有效富集。将产物进行纯化。
图 3 二次PCR产物电泳图
Figure 3. Electrophoresis result of the first and second PCR
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将纯化后的产物与pMD-19T载体连接，并导入大肠杆菌DH5a中。在含Amp的LB固体培养基平板上进行蓝白斑筛选，挑取白色菌落保存在含有Amp的LB液体培养基的96孔细胞培养板中。吸取所有克隆的菌液进行PCR扩增检测，分析消减效率。以GAPDH基因为PCR扩增目的片段(GAPDHF：5-GAGAAGCCAGTCACCATCTT-3、GAPDHR：5-GTTC ACACCCATCACAAACA-3)，如图 4所示。由图 4可知，消减后的GAPDH经28个循环扩增出现条带，而未消减对照经18个循环扩增就出现目的条带，说明经过消减杂交及抑制性PCR扩增，tester和driver中共同转录的基因得到了有效扣除。
图 4 消减cDNA文库消减效率分析
Figure 4. Cutting efficiency analysis of subtracted cDNA library
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图 5是对文库插入片段的检测，经蓝白菌落初步筛选出具有插入系列的克隆有400个，菌落PCR插入效率达80%以上。图 5为随机挑取的12个菌斑所进行的PCR分析结果。在阳性克隆中，PCR扩增片段大小主要集中于200~1 000 bp，说明转化效率高，PCR特异性较好，非特异性扩增现象较少，说明建立的百合花柱抑制消减杂交文库较成功。
图 5 消减cDNA文库部分菌斑分析
Figure 5. Analysis of partial plaque of subtracted cDNA library
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随机挑选180个克隆送去测序，共测出160个，20个克隆未测序出结果。去掉低质量的EST序列，组装之后，得到Contigs 18个、Singlets 120个、Unigenes 138个，冗余序列占序列全部的13.75%，即非冗余序列138条，平均序列469bp。通过BLASTX比对，有13个Contigs和85个Singlets共113条EST序列，与登录在NCBI非冗余蛋白质数据库中的蛋白质序列存在相似性，占有效EST总数的80.71%。
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从差异筛选中筛选出的113个EST序列，有70条可以归类到GO(gene ontology)的相应条目(terms)中。这70条EST序列显著富集于23个GO term中，富集于生物学过程(biological process)、细胞组分(cellular component)、分子功能(molecular function)的分别有5、8、10个GO terms(图 6)。
图 6 试验组和驱动组中差异表达基因的GO分类
Figure 6. GO classification of different expression genes of tester and driver
在细胞组分类别中，序列富集于细胞、细胞部分、高分子配合物、细胞器、细胞器部分；在分子功能类别中，富集于抗氧化剂、结合、催化、电子载体、结构分子、转录调节因子、翻译调节因子、转运；在生物学过程类别中，富集于解剖结构形成、生物调控、细胞组分生物起源、细胞组分组织、细胞过程、建立定位、定位、新陈代谢过程、色素、刺激反应。
对能够注释的基因进行分类，发现它们主要集中在信号转导类基因、转运类基因、抗逆相基因、蛋白合成类相关基因(表 2)。
表 2 试验组与驱动组部分差异表达基因分类
Table 2. Classification of partial different expression of tester and driver
功能类别
Functional category基因注释
Gene annotation基因ID
Gene IDE值
E value信号转导相关基因Signal transduction genes 类钙周期素结合蛋白Ealeyelin- binding protein(CacyBP) OS37、OS41 2×10-66,3×10-63 丝氨酸/苏氨酸蛋白磷酸酶Serine/threonine-protein phosphatase 2A catalytic subunit3(PP2A-3) OS52、OSP27 0，2×10-11 ADP核糖基化因子ADP-ribosylation factor(ARF) OS85 3×10-118 富含亮氨酸重复序列类似受体激酶Leucine-rich repeat receptor kinase (LRR-RLKs) OSP4 3×10-73 周期素依赖性蛋白激酶调控基因Cyclindependent kinase(CDK) OSP12 4×10-20 丝氨酸/苏氨酸蛋白磷酸酶2A调节亚基BSerine/threonine-protein phosphatase 2A catalytic subunitB(PP2A-B) OSP19 7×10-baby5 S期激酶相关蛋白1S-phase kinase associated protein(SKP1) OSP32 8×10-55 小G结合蛋白Small GTP-binding protein(small GTP) OSP62 9×10-3 SCA基因Stigma/style Cysteine-rich Adhesin(SCA) OS36 3×10-12 钙依赖蛋白激酶Calcium-dependent protein kinase(CDPK) OSP63 3×10-3 胚胎发育晚期丰富蛋白1Late embryogenesis abundant proteins 1(LEC1) OSP47 3×10-8 转运类相关基因
Transport-related genes焦磷酸盐能膜质子泵3Pyrophosphate-energized membrane proton pump 3 OS33 8×10-73 质膜内在蛋白1Plasma membrane intrinsic proteins 1(PIP1) OS35、OSP21 2×10-62,1×10-9 酰基载体蛋白Acyl carrier protein(ACP) OS55、OS87 4×10-26,4×10-26 ADP/ATP载体蛋白ADP/ATPcarrier protein(AAC) OS59 5×10-25 水通道蛋白2Aquaporin 2(AQP2) OS109 6×10-74 焦磷酸酶/磷酸二酯酶Nucleotide pyrophosphatase/phosphodiesterase1(ENPP) OSPP57 2×10-45 SCA基因Stigma/style Cysteine-rich Adhesin(SCA) OS36 3×10-12 质膜ATPase 4-likePlasma membrane atpase(PM-ATPase 4L) OSPP06 2×10-66 抗逆防御相关基因Stress/defense genes 亲环蛋白基因Cyclophilin(CyP) OS17 5×10-23 半胱氨酸合酶Cysteine synthase(Csase) OS25 4×10-77 过氧化氢酶2Catalase2(CAT2) OS29 1×10-36 分子伴侣CLPB Molecular chaperone CLPB(CLPB) OS65 1×10-107 抗坏血酸过氧化物酶Ascorbate peroxidase(APX) OS69 8×10-89 脂氧合酶lipoxygenase(LOX) OS71 2×10-10 丝氨酸乙醛酸氨基转移酶Rine:Glyoxylateaminotrans-ferase(SGAT) OS74 4×10-175 细胞色素P450类TBP蛋白Cytochrome P450 like TBP(CYP-TBP) OS82 0 ACC氧化酶ACC oxidase(ACO) OS88 4×10-180 脱水蛋白Dehydrin(DHN) OS90 2×10-5 液泡加工酶3Vacuolar processing enzyme(VPE3) OS95 3×10-60 硫氧还原蛋白Thioredoxin(TRX) OS108 5×10-50 谷胱甘肽过氧化物酶Glutathione peroxidase(GSH-Px) OSP59 2×10-19 金属硫蛋白Metallothionein(MT) OS3、OSP38 2×10-113、3×10-64 蛋白质命运相关基因Protein fate related genes 转录起始因子IIB-like Transcription initiation factor IIB-like(IIB-L) OS119 4×10-42 起始因子iso-4F Eukaryotic initiation factor 4F (eIF-(iso)4F) OS67 4×10-17 翻译起始因子eIF-5A前体蛋白Translation initiation factor 5A precursor(ec-eIF5A) OSP50 6×10-66 核糖体蛋白L2 Ribosomal protein(rp-L2) OS6、OS96 2×10-10,3×10-6 60S核糖体蛋白60S ribosomal protein(RP) OSP7、OSP16 5×10-87,5×10-51 在信号转导类基因中，包含类钙周期素结合蛋白、丝氨酸/苏氨酸蛋白磷酸酶PP2A-3、ADP核糖基化因子、富含亮氨酸重复序列类似受体激酶、周期素依赖性蛋白激酶调控基因、丝氨酸/苏氨酸蛋白磷酸酶2A调节亚基B、SKP1蛋白、小G结合蛋白、SCA基因、钙依赖蛋白激酶、胚胎发育晚期丰富蛋白1。
在转运类相关基因中，包含焦磷酸盐能膜质子泵3、质膜内在蛋白1、酰基载体蛋白、ADP/ATP载体蛋白、水通道蛋白(AQP2)、焦磷酸酶/磷酸二酯酶、SCA基因。
在抗逆相关基因中，包括亲环蛋白基因、半胱氨酸合酶基因、过氧化氢酶、分子伴侣、抗坏血酸过氧化物酶、脂氧合酶、丝氨酸乙醛酸氨基转移酶、细胞色素P450 TBP蛋白、ACC氧化酶、脱水蛋白、液泡加工酶3、硫氧还原蛋白、谷胱甘肽过氧化物酶、金属硫蛋白。
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通过测序后比对的结果及查找基因功能，从70条EST序列中挑选了约25个基因，进行RT-PCR验证，最终筛选出10个基因。由于这10个基因在柱头、花柱、子房和花瓣、叶片、茎、鳞茎中的表达量存在差异，可能影响不亲和性杂交。使用特异性引物分别扩增；使用18S作为内参基因(见图 7)。60S核糖体蛋白在不亲和性杂交花柱中表达量上调；V-H+-ATPase、蛋白磷酸激酶PP2A-3、分子伴侣CLPB、过氧化氢酶CAT2、质膜ATPase、焦磷酸酶质子泵、肌动蛋白解聚合因子等在亲和性杂交花柱中表达量上调；钙依赖蛋白激酶CDPK、小G结合蛋白表达量无明显变化。60S核糖体蛋白、V-H+-ATPase、小G结合蛋白、蛋白磷酸激酶PP2A-3、分子伴侣CLPB、过氧化氢酶CAT2、质膜ATPase和焦磷酸酶质子泵在成熟的花柱(大量分泌液)和非成熟花柱(无分泌液)中存在差异表达。结果证明这8个基因可能对不亲和性杂交有影响。60S核糖体蛋白和V-H+-ATPase在叶片中均无表达或表达量极少；钙依赖蛋白激酶CDPK在花瓣、叶片、茎和鳞茎中无表达或表达量极少，而且在T和D中表达量相似，说明钙依赖蛋白激酶CDPK在雌蕊中表达量均较高，可能受到花粉管的调控，与花粉管互作。
图 7 基因表达量检测
Figure 7. Detection of gene expression level
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本研究发现，百合亲和性杂交与不亲和性杂交的雌蕊之间，存在许多差异表达基因，它们主要集中在信号转导类、抗逆防御类、转运类、蛋白合成类等类别。
前人发现，雌蕊优势或特异表达基因中存在大量信号转导类基因[35, 44]，包括激酶类、蛋白磷酸酶类、信号肽类、泛素介导的信号转导类等。雌蕊中激酶类基因优势表达，可能与雌蕊发育、防御反应、花粉管与雌蕊的信号交流有关[37]。本研究中，差异表达基因中富集许多激酶类基因，推测其可能与雌蕊花粉管之间信号交流有关。雌蕊中的信号肽通过多种形式参与调控花粉管与雌蕊的信号交流[2]。SCA基因是百合雌蕊分泌的信号肽，被认为是一种花粉管粘附因子，与Chemocyanin协同，影响着花粉管在花柱中粘附与定向生长[45]。本研究中，不亲和性杂交后百合雌蕊SCA基因表达下调，可能降低了花粉管在雌蕊花柱道中的粘附与定向生长，从而影响杂交亲合性。青扦(Picea wilsonii)蛋白磷酸酶通过调控囊泡与花粉管顶端的融合及花粉管内外Ca2+的动态变化两方面调控花粉管的生长[46]。本研究中，不亲和性杂交雌蕊中蛋白磷酸酶PP2A-3的表达量下调，可能因此影响了花粉管的正常生长。
植物雌蕊中已发现的优势表达基因，有些属于抗逆防御类[35]。湿柱头授粉受精花粉时，柱头表面会出现富含营养的分泌液，必须有一种抗逆防御机制来保护柱头及分泌液免受病原微生物的侵害，免受氧化、热压迫等危害。在胁迫条件下，分子伴侣可防止蛋白质凝聚、变性、修复变性蛋白，起到稳定蛋白质结构的作用[47]。本研究中，试验组雌蕊中的分子伴侣CLPB显著下调，可能导致其稳定雌蕊中相关信号蛋白质的能力降低，从而导致花粉管伸长受限增强。过氧化氢酶可以清除H2O2，高H2O2会抑制花粉萌发和花粉管伸长[48]。本研究中，试验组雌蕊中的过氧化氢酶基因显著下调，可能导致不能有效地解除H2O2对于花粉管萌发和伸长的抑制。
前人发现，雌蕊优势或特异表达基因中富集大量转运类基因[35, 49]。授粉后，花柱道细胞向花柱道输送营养与信号物质，花粉管从胞外基质中吸收营养或结构物质，均离不开转运类基因的作用。焦磷酸酶质子泵(H+-PPase)，定位于液泡膜及质膜上，能够水解无机焦磷酸，产生自由能，向膜内泵入大量H+，形成跨膜电化学梯度，建立跨膜质子驱动力[50]。花粉管生长需要焦磷酸酶质子泵形成跨膜质子驱动力，进行物质跨膜主动运输。前人研究发现，液泡膜焦磷酸酶质子泵被抑制后，花粉管生长受阻[51]。本研究中，试验组雌蕊中的焦磷酸酶质子泵基因表达下调，暗示花粉管的生长可能因此受阻。
很多核糖体蛋白除具有组成核糖体和参与蛋白质生物合成的功能外，还具有参与复制、转录加工、翻译调控、DNA修复、调控发育、调控细胞凋亡等作用。真核转录起始因子是真核细胞翻译水平上的重要调节因子。翻译起始因子eIF-5A通过其不同异构体的过量表达使细胞走向增殖或者凋亡；还可以通过与核膜上的RNA转运蛋白相互作用，起到调控mRNA运输以及蛋白质翻译方面的作用[52]。本研究中，eIF-5A在亲和性与不亲和性杂交后雌蕊中差异表达，是否与调控花粉管细胞凋亡有关，值得进一步研究。
总之，在东方百合×卷瓣组野生百合不亲合性杂交后的雌蕊，与亲和性杂交后的雌蕊相比，差异表达基因主要集中在信号转导、抗逆防御、转运、蛋白命运调控等功能类别。据此推测，不亲和性杂交后，可能引起雌蕊与花粉管信号交流异常、物质转运能力减弱、雌蕊耐受逆境能力降低，从而导致杂交不亲和性。
Analysis of different expressiongenes between cross-compatibility and cross-incompatibility within pistils of Lilium spp
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摘要: 东方百合与卷瓣组野生百合杂交(简称OS组合)，属于亲缘关系最远的组间杂交，杂交障碍最大。挖掘雌蕊中杂交亲和性与不亲和性差异表达基因，有可能解释不亲和性杂交中引起花粉管定向生长异常的机理，进而阐明不亲和性杂交的分子机理，为实施克服杂交不亲和障碍技术措施提供依据。本研究采用抑制消减杂交技术(SSH技术)，分别以不亲和性杂交和亲和性杂交的东方百合雌蕊为试验组(tester)和驱动组(driver)，建立正向抑制消减杂交文库，随机挑选180个阳性克隆，经过测序、去劣、去冗余、序列比对，有113个EST与数据库中的已知序列具有同源性，最终获得10个差异表达基因。采用RT-PCR技术对10个差异表达基因进行验证，其中有1个基因在不亲和性杂交的雌蕊中表达上调、7个基因表达下调、2个基因无明显变化。对差异表达基因进行功能注释及分类，差异表达基因主要集中于信号转导(包括丝氨酸/苏氨酸蛋白磷酸酶PP2A-3、钙依赖蛋白激酶CDPK、小G结合蛋白)、抗逆防御(包括分子伴侣(clpB)、过氧化氢酶CAT2)、转运(包括质膜ATPase、焦磷酸酶质子泵)、蛋白命运(包括60S核糖体蛋白)等功能类别。据此推测，在东方百合×山丹组(系)间不亲合性杂交育种过程中，可能引起雌蕊与花粉管信号交流异常、物质转运能力减弱和雌蕊耐受逆境能力降低，从而导致杂交不亲和性。Abstract: The hybridization of Lilium Oriental hybrids and wild lily of Sect. Sinomartagon Comber is the farmost genetic relationship and it's difficult to achieve. Researching the different expression genes in pistil of cross-compatibility and cross-incompatibility may explain the mechanism of causing orientated growth of pollen tube and the molecular mechanism of incompatible hybridization, and provide the basis for overcoming cross-incompatibility. We constructed a forward suppression subtractive hybridization (SSH) library by using the cross-incompatible Lilium Oriental hybrid pistils as the tester, and the cross-compatible ones as the driver. 180 positive clones were randomly screened out. Through sequencing, deleting the bad, removing the redundant and aligning the sequences, 113 ESTs were found to be homologous with known sequences in the bioinformatics databases. RT-PCR was performed to test the 10 selected different expression genes, which showed 1 gene was up-regulated, 7 genes were down-regulated and 2 genes were almost unchanged. Gene annotation and functional category analysis showed that the different expression genes mainly contained signal transduction(including PP2A-3, CDPK and small GTP-binding protein), transport-related(including clpB and CAT2), stress/defense(including ATPase and pyrophosphatase proton pump), protein fate related genes(including 60S ribosomal protein). It was speculated that the incompatibility in the section cross of Oriental hybrids ×Lilium pumilum was probably resulted from the abnormal signal communication between pistils and pollens, the deduced substance transport ability in pistils, the weaker stress resistance ability in pistils after incompatible hybridization.
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图 1 总RNA和mRNA电泳检测
M. DL 200 marker; T.试验组; D.驱动组。
Figure 1. Electrophoresis detection of total RNA and mRNA
M, DL 200 marker; T, Tester; D, Driver.
图 2 cDNA酶切前后的电泳图
M. DL 2 000 marker; T1.试验组cDNA; T2.酶切后试验组cDNA; D1.驱动组cDNA; D2.酶切后试验组cDNA。
Figure 2. Electrophoresis pattern of cDNA before and after digestion
M, DL 2 000 marker; T1, Tester cDNA; T2, Tester cDNA after digestion; D1, Driver cDNA; D2, Driver cDNA after digestion.
图 3 二次PCR产物电泳图
M.DL 2 000 marker；E1.正向消减第一次PCR产物; 1-c.正向未消减第一次PCR产物；pE1.正向消减第二次PCR产物; p1-c.正向未消减第二次PCR产物。
Figure 3. Electrophoresis result of the first and second PCR
M, DL 2 000 marker; E1, first PCR production of positive subtraction; 1-c, first PCR production of positive non-subtraction; pE1, second PCR production of positive subtraction; p1-c, second PCR production of positive non-subtraction.
图 4 消减cDNA文库消减效率分析
M.DL 2 000 marker；1、2、3、4、5分别为消减后第18、23、28、33、36个循环的PCR产物；6、7、8、9、10分别为未消减的第18、23、28、33、36个循环的PCR产物。
Figure 4. Cutting efficiency analysis of subtracted cDNA library
M, DL 2 000 marker; 1, 2, 3, 4, 5 are the PCR production of the 18th, 23rd, 28th, 33rd, 36th cycles after subtracted respectively; 6, 7, 8, 9, 10 are the non-subtractive PCR production of the 18th, 23rd, 28th, 33rd, 36th cycles, respectively.
图 6 试验组和驱动组中差异表达基因的GO分类
细胞cell；细胞组分Cell component；高分子配合物Macromolecular complex；细胞器Organelle；细胞器组分Organelle part；抗氧化剂Antioxidant；结合Binding；催化剂Catalytic；电子载体Electron carrier；结构分子Structural molecule；转录调节因子Transcription regulator；转运Transporter；解剖结构组成Anatomical structure formation；生物调节Biological regulation；细胞组成生物源Cellular component biogenesis；细胞组织组件Cellular component organization；细胞过程Cellular process；建立定位Establishment of localization；定位Localization；代谢过程Metabolic process；色素形成Pigmentation；刺激反应Response to stimulus
Figure 6. GO classification of different expression genes of tester and driver
图 7 基因表达量检测
T.试验组Tester; D.驱动组Driver; 1.柱头Stigma；2.花柱Style；3.子房Ovary；4.花瓣Petal；5.叶Leaf；6.茎Stem；7.鳞茎Bulb
Figure 7. Detection of gene expression level
表 1 特异性引物
Table 1. Specific primer
基因名称
Gene name上游引物(5′-3′)
Forward primer(5′-3′)下游引物(5′-3′)
Reverse primer(5′-3′)退火温度
Annealing
temperature/℃60S核糖体蛋白
60S ribosomal protein(RP)AATCCCTTGAATCCTCTTGCC AGAGAAGGCGAAGATGGTG 54 液泡膜H+-ATPase
Vacuolar H+-ATPase(V-H+-ATPase)AGAGAGAAGATGACCTGAATGAAAT CCAAACGATGCTTGATGACG 54 小G结合蛋白
Small GTP-binding protein (small GTP)ACCTGCTCAAGAACTAGAAG AGTAGGGAACAAAACAAACAC 51 丝氨酸/苏氨酸蛋白磷酸酶
Serine/Threonine-protein phosphatase 2A catalytic subunit3(PP2A-3)GGAGGAAAAGATGAGCGGG AATAATAGCCACGGTCCACATAATC 55 分子伴侣CLPB
Molecular chaperone CLPB(CLPB)TGACTATGCTGTTGATCTGC ATGCAGTTTCGAGATTGAT 49 过氧化氢酶2
Catalase2(CAT2)CAACCTGGAGAGCGATACC GTCGAAGGTAGTAAGCC 50 质膜ATPase
Plasma membrane atpase (PM-ATPase)AGTTTAATGCAAGCGATAT AGCAAGAAGAATTATGGG 48 焦磷酸盐能膜质子泵3
Pyrophosphate-energized membrane
proton pump 3AATCCCTTATTCCACAAACAAG ATCTTCGTTGACCTGGCTAAG 52 肌动蛋白解聚合因子
Actin depolymerizing factor(ADF)GTACCCAAACAAGAAGCACAT TGATGTGTCGAGGGTGAGGAGT 53 钙依赖蛋白激酶Calcium-dependent protein kinase(CDPK) CAGTATTAGAACTCATTGGCAC GTAATCCCCATATTCACTGCTG 54 
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表 2 试验组与驱动组部分差异表达基因分类
Table 2. Classification of partial different expression of tester and driver
功能类别
Functional category基因注释
Gene annotation基因ID
Gene IDE值
E value信号转导相关基因Signal transduction genes 类钙周期素结合蛋白Ealeyelin- binding protein(CacyBP) OS37、OS41 2×10-66,3×10-63 丝氨酸/苏氨酸蛋白磷酸酶Serine/threonine-protein phosphatase 2A catalytic subunit3(PP2A-3) OS52、OSP27 0，2×10-11 ADP核糖基化因子ADP-ribosylation factor(ARF) OS85 3×10-118 富含亮氨酸重复序列类似受体激酶Leucine-rich repeat receptor kinase (LRR-RLKs) OSP4 3×10-73 周期素依赖性蛋白激酶调控基因Cyclindependent kinase(CDK) OSP12 4×10-20 丝氨酸/苏氨酸蛋白磷酸酶2A调节亚基BSerine/threonine-protein phosphatase 2A catalytic subunitB(PP2A-B) OSP19 7×10-baby5 S期激酶相关蛋白1S-phase kinase associated protein(SKP1) OSP32 8×10-55 小G结合蛋白Small GTP-binding protein(small GTP) OSP62 9×10-3 SCA基因Stigma/style Cysteine-rich Adhesin(SCA) OS36 3×10-12 钙依赖蛋白激酶Calcium-dependent protein kinase(CDPK) OSP63 3×10-3 胚胎发育晚期丰富蛋白1Late embryogenesis abundant proteins 1(LEC1) OSP47 3×10-8 转运类相关基因
Transport-related genes焦磷酸盐能膜质子泵3Pyrophosphate-energized membrane proton pump 3 OS33 8×10-73 质膜内在蛋白1Plasma membrane intrinsic proteins 1(PIP1) OS35、OSP21 2×10-62,1×10-9 酰基载体蛋白Acyl carrier protein(ACP) OS55、OS87 4×10-26,4×10-26 ADP/ATP载体蛋白ADP/ATPcarrier protein(AAC) OS59 5×10-25 水通道蛋白2Aquaporin 2(AQP2) OS109 6×10-74 焦磷酸酶/磷酸二酯酶Nucleotide pyrophosphatase/phosphodiesterase1(ENPP) OSPP57 2×10-45 SCA基因Stigma/style Cysteine-rich Adhesin(SCA) OS36 3×10-12 质膜ATPase 4-likePlasma membrane atpase(PM-ATPase 4L) OSPP06 2×10-66 抗逆防御相关基因Stress/defense genes 亲环蛋白基因Cyclophilin(CyP) OS17 5×10-23 半胱氨酸合酶Cysteine synthase(Csase) OS25 4×10-77 过氧化氢酶2Catalase2(CAT2) OS29 1×10-36 分子伴侣CLPB Molecular chaperone CLPB(CLPB) OS65 1×10-107 抗坏血酸过氧化物酶Ascorbate peroxidase(APX) OS69 8×10-89 脂氧合酶lipoxygenase(LOX) OS71 2×10-10 丝氨酸乙醛酸氨基转移酶Rine:Glyoxylateaminotrans-ferase(SGAT) OS74 4×10-175 细胞色素P450类TBP蛋白Cytochrome P450 like TBP(CYP-TBP) OS82 0 ACC氧化酶ACC oxidase(ACO) OS88 4×10-180 脱水蛋白Dehydrin(DHN) OS90 2×10-5 液泡加工酶3Vacuolar processing enzyme(VPE3) OS95 3×10-60 硫氧还原蛋白Thioredoxin(TRX) OS108 5×10-50 谷胱甘肽过氧化物酶Glutathione peroxidase(GSH-Px) OSP59 2×10-19 金属硫蛋白Metallothionein(MT) OS3、OSP38 2×10-113、3×10-64 蛋白质命运相关基因Protein fate related genes 转录起始因子IIB-like Transcription initiation factor IIB-like(IIB-L) OS119 4×10-42 起始因子iso-4F Eukaryotic initiation factor 4F (eIF-(iso)4F) OS67 4×10-17 翻译起始因子eIF-5A前体蛋白Translation initiation factor 5A precursor(ec-eIF5A) OSP50 6×10-66 核糖体蛋白L2 Ribosomal protein(rp-L2) OS6、OS96 2×10-10,3×10-6 60S核糖体蛋白60S ribosomal protein(RP) OSP7、OSP16 5×10-87,5×10-51
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