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减数分裂是植物生活史中的一个重要环节,是有性生殖的前提,是保持物种稳定性的基础[1]。本研究的实验材料‘无籽’瓯柑Citrus suavissima ‘Seedless’是1996 年发现的一个瓯柑Citrus suavissima的无核突变体,具有果实完全无核的特性[2]。张迟等[3]采用半薄树脂切片、扫描电镜、透射电镜等方法对其花药发育过程和花粉形态进行观察,发现在四分体时期出现异常,在单核期出现明显异常,认为花粉败育开始于小孢子母细胞减数分裂期至四分体时期。近年来,模式植物减数分裂行为中的染色体配对和联会方面的研究取得了较大进展。PANOLI等[4]研究了拟南芥Arabidopsis thaliana的直系同源基因Atspo11-1,染色体重组起始于Spoll调控的DNA双链解开,Atspo11-1突变体中Atspo11-1的缺失会抑制染色体破碎化。LI等[5]利用荧光原位杂交技术观察了拟南芥突变体rad51c-1的早期减数分裂行为,发现该突变体的同源染色体无法正常配对并出现严重的破碎化现象。敲除RAD51C基因后得到的不育植株伴随有大孢子和花粉母细胞粗线期染色体异常[6]。ITO等[7]研究了拟南芥中的MALE STERILITY1(MS1)基因,它对减数分裂后的花粉发育很重要,与花粉壁、花粉细胞质内容物和绒毡层发育有关。在果树方面,目前的研究多采用分子标记法来发掘育性相关基因。如王跃进等[8]通过分离、提取和纯化经随机扩增多态DNA(RAPD)标记获得的特异片段,根据其序列人工合成一条寡聚核苷酸作引物(5′CCAGT TCGCC CGTAA ATG 3′),在多个葡萄Vitis vinifera品系中进行验证,凡出现约590 bp的DNA片段者即为葡萄无核基因携带者和无核性状表现者。杨英军等[9]又进一步根据此特异片段设计合成了2对引物,开发出鉴别无核品种的SCAR(特定序列扩增标记)。姚春潮等[10]利用 RAPD 技术对美味猕猴桃Actinidia deliciosa var. deliciosa海瓦德×秦雄 201 杂交 F1代雌雄分离群体进行分析,通过对 300 个随机引物的筛选和研究,得到了与猕猴桃雄性基因链锁的 RAPD 标记S1032-850。NWAFOR等[11]采集了野生型葡萄和一个无核突变体材料的果实进行转录组测序技术(RNA-Seq)分析,对得到的差异表达基因进行表达模式和功能富集分析,发现花粉和胚囊两者的发育途径具有密切关联,但无核性状具体由哪些基因控制仍不明了。本研究根据‘无籽’瓯柑及其野生型小孢子母细胞时期花药转录组的测序结果,选择了RAD51和MS1这2个减数分裂特性基因,研究它们在‘无籽’瓯柑和瓯柑花粉发育不同时期的相对表达量差异,来分析‘无籽’瓯柑小孢子母细胞减数分裂异常的分子机制。此外,鉴于减数分裂只在特定时期和特定部位发生,本研究选取了花蕾和花药2种材料进行基因相对表达量的测定,进而分析不同材料所得结果的差异。
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供试材料瓯柑和‘无籽’瓯柑采自丽水市林业科学研究院百果园。当花蕾刚露白时开始取样,根据预备试验的结果,采集‘无籽’瓯柑和瓯柑花粉母细胞形成期(Ⅰ),四分体时期(Ⅱ),单核花粉粒时期(Ⅲ),双核花粉粒时期(Ⅳ) 和花粉粒成熟期(Ⅴ)共5个时期的花蕾和对应花药,5个时期的花蕾直径分别为2.0~2.4 mm,2.8~3.1 mm,3.5~4.5 mm,4.5~6.5 mm和即将开放的花蕾(6.5~7.1 mm,图 1),保存于-70 ℃超低温冰箱。
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采用改良的Trizol法提取瓯柑和‘无籽’瓯柑各5个时期花蕾的RNA,用Aidlab公司的‘EASYspin PLUS植物RNA快速提取试剂盒’提取花药RNA,10.0 g·kg-1的琼脂糖凝胶对各样品的 RNA进行电泳检测,再经NanoDrop-1000测吸光度值,检测样品RNA的质量。
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cDNA的合成参照TaKaRa反转录试剂盒PrimeScrip® RT reagent kit with gDNA Eraser (perfect real time)的说明指南。根据转录组测序结果中的基因序列(已提交GenBank)设计荧光定量特异引物(表 1),以甜橙Citrus sinensis的Actin基因(GU911361)为内参,采用定量仪器CFX96 real-time system实时荧光定量PCR仪(Bio-Rad)和荧光染料SYBR®Premix Ex TaqTM Ⅱ(Tli RNaseH Plus)进行实时表达量测定。反应程序为95 ℃,30 s;95 ℃,5 s;57 ℃,30 s,39个循环;65~95 ℃,隔5 s上升0.5 ℃做融解曲线。设立重复3个·样品-1,根据2-△△Ct法[12]进行基因相对表达量计算,利用SPSS 16.0的最小显著差(LSD)法在0.01水平上比较2个基因在不同组织和不同时期中的表达量差异。
表 1 实时荧光定量聚合酶链式反应(qRT-PCR)特异引物
Table 1. Specific primers for qRT-PCR
基因 登录号 正向引物(5'→3') 反向引物(5'→3') RAD51C KU204895 AGAAGCTGAAAGATGCGGGT GATGGAGTTGGGTAGCACTGG MS1 KU204897 AGTGCTTGCATTGATGATCC TGAGGCATTTAGGCATAACG 内参 Actin GU911361 ATCTGCTGGAAGGTGCTGAG CCAAGCAGCATGAAGATCAA
Expression of microsporocyte meiosis with special genes RAD51 and MS1 in Citrus suavissima ‘Seedless’
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摘要: ‘无籽’瓯柑Citrus suavissima ‘Seedless’是野生型瓯柑Citrus suavissima的芽变,其花粉完全败育,败育起始于小孢子母细胞减数分裂形成四分体时期。研究‘无籽’瓯柑小孢子母细胞减数分裂异常的分子机制,有利于深入认识果树芽变的发生机制。随机选择“无籽”瓯柑和瓯柑各3株,根据花蕾直径采集花粉母细胞时期(Ⅰ),四分体时期(Ⅱ),单核花粉粒时期(Ⅲ),双核花粉粒时期(Ⅳ)及花粉粒成熟期(Ⅴ)共5个时期的花蕾并分离花药,利用实时荧光定量聚合酶链式反应(qRT-PCR)分析减数分裂特性基因RAD51和MS1的相对表达量,3次重复。通过SPSS 16.0的最小显著差法(LSD)在0.01水平上进行各样品间的相对表达量差异分析。结果表明:RAD51和MS1基因的表达高峰均集中在第Ⅰ时期和第Ⅱ时期,且此时花药中的相对表达量均极显著(P<0.01)高于花蕾。第Ⅰ时期花药中,‘无籽’瓯柑RAD51的相对表达量为瓯柑的3.7倍,MS1可达300倍;但同时期花蕾中,RAD51的相对表达量仅1.1倍,MS1为140.0倍。‘无籽’瓯柑RAD51基因在第Ⅰ时期和第Ⅱ时期的相对表达量极显著(P<0.01)高于瓯柑,说明‘无籽’瓯柑小孢子母细胞和四分体时期的细胞内均存在DNA受损现象;‘无籽’瓯柑MS1基因在第Ⅰ时期的相对表达量极显著(P<0.01)高于瓯柑,但第Ⅱ时期极显著(P<0.01)低于瓯柑,推测‘无籽’瓯柑败育花粉的形成与MS1基因表达紊乱引起油脂分泌、转运异常密切相关。图6表1参24Abstract: To obtain the relative expression of two meiosis special genes——RAD51 and MS1 (Male Sterility1),Citrus suavissima ‘Seedless’, a bud variation of its wild type Ougan (Citrus suavissima) with completely aborted pollens that appear abnormally from the tetrad stage, were studied through the molecular mechanism of abnormal meiosis. According to flower bud diameter, five stages of flower buds and anther——pre-meiotic (PM, I), tetrad (T, II), single-celled (SC, III), dual-core (DC, IV), and mature pollen (MP, V)——were collected and each was an composite sample from three trees. An experiment in real-time quantitative polymerase chain reaction (qRT-PCR) was conducted by three replications in each sample and the method of least significant difference(LSD) with SPSS 16.0 was used to test the expression differences between samples under the level of P<0.01. Results showed that expression of RAD51 at Stage I of the anther and the flower buds for C. suavissima ‘Seedless’ was significantly higher (P<0.01) than Ougan. At Stage I of the anther, the relative expression of RAD51 in C. suavissima ‘Seedless’ was 3.7 times higher than in Ougan with MS1 reaching about 300 times higher. However, at Stage I of the flower buds, RAD51 was only 1.1 times higher and MS1 was about 140 times higher. The significantly higher expression of RAD51 at Stage I indicated that a phenomenon of DNA damage might exist in the microsporocyte and tetrad periods of C. suavissima ‘Seedless’; for gene MS1, significantly higher expression of C. suavissima ‘Seedless’ for Stage I also meant that the aborted pollens of C. suavissima ‘Seedless’ had a relationship with the abnormal secretion and transfer of oil lipids that were influenced by disordered expression of MS1.[Ch, 6 fig. 1 tab. 24 ref.]
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Key words:
- horticulture /
- Citrus suavissima ‘Seedless’ /
- meiosis /
- flower buds /
- anther /
- RAD51 /
- MS1 /
- qRT-RCR
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表 1 实时荧光定量聚合酶链式反应(qRT-PCR)特异引物
Table 1. Specific primers for qRT-PCR
基因 登录号 正向引物(5'→3') 反向引物(5'→3') RAD51C KU204895 AGAAGCTGAAAGATGCGGGT GATGGAGTTGGGTAGCACTGG MS1 KU204897 AGTGCTTGCATTGATGATCC TGAGGCATTTAGGCATAACG 内参 Actin GU911361 ATCTGCTGGAAGGTGCTGAG CCAAGCAGCATGAAGATCAA -
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https://zlxb.zafu.edu.cn/article/doi/10.11833/j.issn.2095-0756.2016.06.001