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杨属(Populus L.)植物分布广泛,不仅具有较高的经济和生态价值,同时也是木本模式植物,在林木分子生物学研究等方面具有重要作用。然而,作为北方城市重要的道路绿化和城市景观树种,杨树在提高城市绿化水平的同时,也因其花粉在每年春季大量飘散而引起了一定的植源性污染问题。杨树雄株散落的花粉以空气为传播媒介,通过人体呼吸及皮肤接触造成呼吸道疾病和皮肤过敏等问题,危害着人体健康[1-3]。除更换树种外,目前关于花粉污染的应对策略主要还包括人工喷水增湿、增强地表对花粉的吸附能力等。另外,日益发展的生物技术推动了林木遗传育种进程,有望成为解决花粉等植源性污染的有效途径之一。目前关于杨树的研究主要聚焦于其材性及抗逆性等方面,而对杨树的生殖发育研究相对较少。
杨树童期较长且雌雄异株,不同于大多数由萼片、花瓣、心皮(雌蕊/雄蕊)、胚珠4轮结构组成的被子植物花,杨树的雌花和雄花均高度简化,其中,雄花着生于苞片腋间,由退化的盘状花被和雄蕊构成,花药为黄色或红色,一般情况下药隔无明显突出[4]。花药为四孢型,成熟的花药壁由一层表皮、一层内层、两层中层和单层绒毡层组成。表皮宿存,内层发育成纤维状增厚,中层是短命的,其中一层在小孢子阶段变为扁平,最后被挤碎,另一层则在花药开裂前解体;绒毡层细胞早期是单核的,而当花粉母细胞减数分裂时则成为双核或多核的,它们在花粉单核时期开始退化,至双核期消失[5]。杨树性别特异的花器官发育是一个复杂的过程,但与其他被子植物花发育类似,杨树的花发育也分为开花诱导、花的发端和花器官发育3个受众多基因调控的复杂过程,且它们具有相似的花器官特性基因[6]。
随着研究的不断深入,花器官形成的相关理论逐步完善,由最初的“ABC”模型假说发展成为“ABCDE”模型及四聚体模型,其中,A类和E类基因决定花萼特征,A、B和E类基因一起调控花瓣发育,B、C和E类基因共同决定雄蕊发育,C类和E类基因决定心皮发育,D类和E类决定胚珠发育[7-8]。杨树花发育的相关器官特异性基因的作用不全同于其他开花植物,近年来调控杨树花器官发育的多个ABCDE类基因功能被解析,如:A类基因中的AP1同源基因PTM1、PTM2和B类基因中的AP3同源基因,它们在杨树雌/雄株中的表达有所差异,可能参与了杨树的性别决定[9-10];B类基因中的PI的同源基因PdPI和C类基因PTAG1和PTAG2,与杨树花发育密切相关并且可能影响营养器官的形成和发育[6, 11-12];E类基因中的PTM3、PTM4及PTM6,在杨树雌/雄株中花发育过程中持续表达,在胚珠、花药原基中有着非常高的表达量[13]。
AGAMOUS (AG)是MADS-box基因中研究较为充分的一类基因,属于植物特有的MIKC型C类MADS-box基因,包括euAG/PLENA (PLE)谱系和AGAMOUS-like11(AGL11)谱系,可与其他基因互作共同控制雄蕊及心皮的发育[8, 14-17]。在拟南芥中,AG可控制拟南芥花发育后期的花粉形成[18-20]。另有研究表明,在拟南芥AG突变体的花器官四轮结构中出现了第三轮结构(雄蕊)转化为花瓣、第四轮结构(心皮)发育成一个新的花的情况,而第一、二轮的花结构(萼片及花瓣)与野生型拟南芥花器官并无差异[21-23]。另外,抑制AG会导致菠菜雄性不育[24]。而AGAMOUS (AG)及其同源基因在单性花发育的研究中鲜有报道,在毛果杨雌株中,AG(PtAG1及PtAG2)和STK(AGL11)基因在杨树种毛发育中表现出较强的功能保守性,共同抑制种毛的形成[12],而该基因在杨树雄花花发育中的作用尚无报道。
本研究基于AG2在被子植物花器官的决定作用,对银腺杨‘84K’(Populus alba × P. glandulosa ‘84K’)PagAG2基因时空特异性进行了检测,为进一步研究其在杨树雄花花发育中的功能,进而有针对性地对杨树生殖发育进行调控奠定基础。
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银腺杨‘84K’(Populus alba × P. glandulosa ‘84K’)花序于2019年2月22日采自北京市中国林科院内(115.7°~117.4° E,39.4°~41.6° N),组培材料生长于温度25℃、光强2300 Lx、光周期16 h/8 h的组织培养间中;RNA提取试剂盒购自Aidlab公司(北京);Premix TaqTM酶购、反转录试剂盒、qRT-PCR试剂盒均购自Takara公司(日本);固定液由50%FAA固定液100 mL、无水乙醇50 mL、37%甲醛溶液10 mL、冰乙酸5 mL加DEPC·H2O定容至100 mL;RNA预杂交液/杂交液由20 × SSC 30 mL、50 × Denhardt's 10 mL、10%SDS 5 mL、10 mg· mL−1Salmon DNA 1 mL、Formamide(甲酰胺)50 mL、ddH2O 4 mL,充分混匀后使用0.45 μm滤膜去杂质后使用;抗荧光淬灭封片剂购自生工生物公司(上海)。
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取‘84K’杨越冬花枝,2019年2月22日置于温室水培(0 d),每日更换一次水。对水培后1周内的花序连续采样(0、3、4、5、6、7 d),置于PBS固定液中立即真空抽气固定,并依次完成脱水、透明、浸蜡、包埋、修块、切片、粘片、脱蜡步骤,以番红—固绿法染色[25]。染色完成染色后,在光学显微镜(OLYMPUS公司(BX51)产品)下观察、拍照记录。
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分别提取‘84K’杨组培苗的根、茎、叶及水培后2周内的花序连续采样(水培后0、3、4、5、6、7 d的花序)的总RNA,并反转录合成cDNA。使用荧光定量PCR仪(Roche公司(Light Cycle 480Ⅱ)产品)进行qRT-PCR反应,反应体系为cDNA模板2 μL,Primer-F(10 μmol·L−1) 0.8 μL,Primer-R(10 μmol · L−1) 0.8 μL,XTB Green Premix TaqII(TliRNaseH Plus,2 × )10 μL,ddH2O补足至20 μL。其扩增条件为预变性95℃,30 s;变性95℃,5 s,退火60℃,30 s,40个循环;熔解曲线为95℃ 5 s,65℃ 1 min。以Actin为内参基因,每个样品进行3次重复,用2−ΔΔCT算法进行分析[26],引物见表1。
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基于碱基互补的原则,利用荧光原位杂交技术将细胞原位杂交技术和荧光技术有机结合,用荧光素标记的已知外源RNA作探针,与‘84K’杨雄花组织切片杂交,与待测核酸的靶序列专一性结合,通过检测杂交位点荧光来显示PtAG2核苷酸序列在‘84K’杨雄花中的位置。根据待检测目的基因序列,采用Primer Premier 6.0软件设计,并用探针合成仪器(UNICORN 5.31,沃特曼)合成RNA探针(表2),荧光素标记反义RNA核酸探针,以正义RNA探针为阴性对照。将‘84K’杨雄花序样品放入配制好的固定液(50%FAA固定液100 mL、无水乙醇50 mL、37%甲醛溶液10 mL、冰乙酸5 mL,加DEPC·H2O定容至100 mL)中固定12 h后经梯度酒精脱水后浸蜡、包埋、切片(SQ2125,徕克公司)、摊片(PPTHK-21B,徕克)、捞片,62℃烤箱烤片2 h。石蜡切片脱蜡后,将切片于修复液中煮沸10~15 min,自然冷却。滴加蛋白酶K(20 ug· mL−1)37℃消化,纯水冲洗后PBS洗3次,每次5 min。滴加预杂交液,37℃于杂交仪(S500-24,ThermoBrite)孵育1 h。倾去预杂交液,滴加探针杂交液,置于恒温箱57℃度杂交过夜。洗去杂交液,2×SSC,37℃洗10 min,1×SSC,37℃洗2次,每次5 min,0.5×SSC室温洗10 min。切片滴加DAPI染液,避光孵育8 min,冲洗后滴加抗荧光淬灭封片剂(Anti-Fade Mounting Medium,生工)封片,置于荧光显微镜下(CX41,OLYMPUS)观察并采集图像(紫外激发波长330~380 nm,发射波长420 nm,发蓝光;FAM(488)绿光激发波长465~495 nm,发射波长515~555 nm,发绿光;CY5红光激发波长510~560 nm,发射波长590 nm,发红光)。所有试剂、仪器均需DEPC处理。
表 1 qRT-PCR表达分析引物
Table 1. Primers used in qRT-PCR analysis
基因 Gene 产物长度 Amplification Length/bp 引物序列 Primer sequence 用途 Usage Actin 195 F: 5′-AAACTGTAATGGTCCTCCCTCCG -3′ Internal
referenceR: 5′-GCATCATCACAATCACTCTCCGA -3′ PagAG2 270 F: 5′-GCTTCTCCGAGCAAAGGTCT-3′ qRT-PCR R: 5′-GCAGGCGGCAATCCATTAAC -3′ 表 2 RNA探针序列
Table 2. RNA probe sequences
基因 Gene 序列 Sequence 荧光标记 Fluorescence labeling PagAG2 5'-AUUAUCUGUUCUUUGCGAUGCUGAGGUUGC-3' 5’CY5修饰 红色
(阴性对照)5'-UAAUAGACAAGAAACGCUACGACUCCAACG-3' 5’FAM修饰 绿色
银腺杨PagAG2基因的时空表达分析
Spatiotemporal Expression Analysis of PagAG2 Gene in Populus alba × P. glandulosa '84K'
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摘要:
目的 研究杨树AG类基因(PtAG1及PtAG2)在杨树雄花发育过程中的作用,为杨树分子育种提供基础。 方法 以‘84K’杨(Populus alba × P. glandulosa ‘84K’)花芽为材料,对花芽发育过程连续采样,观察表型发育状况与测定PagAG2基因部位与表达量,通过石蜡切片、实时定量PCR、原位杂交等技术手段,对银腺杨‘84K’(Populus alba × P. glandulosa ‘84K’)PagAG2基因时空特异性进行研究,分析PagAG2基因表达模式。 结果 在‘84K’杨雄花发育过程中,花药逐渐成熟,在此过程中,PagAG2的表达量先增加后降低,并且在‘84K’杨组培苗根、茎、叶中也能够检测到该转录物。原位杂交结果显示:PagAG2在‘84K’杨雄花花药中表达,但在包围花药的周围组织中不表达。 结论 ‘84K’PagAG2基因与‘84K’杨雄花花发育密切相关,有望作为基因工程改良杨树花粉的目标基因用于研究。但PagAG2是否参与杨树其他组织的生长发育调控,在相关育种工作中需要对其做进一步的基因功能分析。 Abstract:Objective To identify the role of PtAG2 gene in the development of male flowers of poplar. Method Based on the regulation of AG genes (PtAG1 and PtAG2) in the stamens and carpels of monoecious plants, the spatial and temporal specificity of PagAG2 gene of Populus alba × P. glandulosa '84K' was studied by means of paraffin section, real-time quantitative PCR and in situ hybridization. Taking the flower branches of P. alba × P. glandulosa '84K' as test materials, the inflorescences were continuously sampled within one week after hydroponic culture to observe the development of male flowers by paraffin sectioned technique. Real-time quantitative PCR was used to detect the expression of PagAG2 in the roots, stems, leaves and inflorescence of P. alba × P. glandulosa '84K'. In situ hybridization was used to detect the specific expression sites of PagAG2 in the flower organs of P. alba × P. glandulosa '84K' male flowers, so the expression pattern of PagAG2 gene was analyzed. Result During the development of male flowers of P. alba × P. glandulosa '84K', the anthers matured gradually. At the same time, the expression of PagAG2 increased at initial and then decreased, and the transcription was also be detected in the roots, stems and leaves of tissue cultured P. alba × P. glandulosa '84K'. The results of in situ hybridization showed that PagAG2 was expressed in the anthers of P. alba × P. glandulosa '84K' male flowers but not in the surrounding tissues. Conclusion PagAG2 gene is closely related to the male flower development of P. alba × P. glandulosa '84K', so it is expected to be a target gene for genetic engineering to improve poplar pollen. However, whether the PagAG2 gene participates in the regulation of the growth and development of other tissues of poplar needs to be further studied. -
Key words:
- Populus alba × P. glandulosa '84K'
- / pollen
- / PagAG2
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表 1 qRT-PCR表达分析引物
Table 1. Primers used in qRT-PCR analysis
基因 Gene 产物长度 Amplification Length/bp 引物序列 Primer sequence 用途 Usage Actin 195 F: 5′-AAACTGTAATGGTCCTCCCTCCG -3′ Internal
referenceR: 5′-GCATCATCACAATCACTCTCCGA -3′ PagAG2 270 F: 5′-GCTTCTCCGAGCAAAGGTCT-3′ qRT-PCR R: 5′-GCAGGCGGCAATCCATTAAC -3′ 表 2 RNA探针序列
Table 2. RNA probe sequences
基因 Gene 序列 Sequence 荧光标记 Fluorescence labeling PagAG2 5'-AUUAUCUGUUCUUUGCGAUGCUGAGGUUGC-3' 5’CY5修饰 红色
(阴性对照)5'-UAAUAGACAAGAAACGCUACGACUCCAACG-3' 5’FAM修饰 绿色 -
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