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早花相关基因HbFT1和HbFT2转化橡胶树的研究

花匠小妙招
2025-11-27 14:37

天然橡胶是重要的工业原料，主要产自巴西橡胶树（Hevea brasiliensis）[1]，其经济生命周期为30~35 a[2]。与许多木本树种一样，橡胶树在开始开花之前有一个5~6 a的幼年期（未成熟期），需要28 a以上的时间来繁殖和选择一个新的无性系用于天然橡胶的商业生产[1,3]，未成熟期是影响短时间内培育新橡胶树的一个严重的制约因素，橡胶树开花调控途径的研究对提高育种效率至关重要。

植物的生长和发育，特别是开花的诱导，受内源和环境信号关键调控因子的严格控制，一般分为光周期途径、温度途径、春化途径、年龄途径、赤霉素途径和自主途径等6条主要途径[4-5]。这些途径并不是孤立存在的，而是通过各种信号途径最终调控整合FLOWERING LOCUS T（FT）、LEAFY（LFY）和SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1（SOC1）等基因实现成花转变的调控[6-7]。FT基因是PEBP家族成员之一，作为成花素，在光周期调控途径中，长日照条件下，受CONSTANS（CO）正调控的FT蛋白从叶片运输到茎端分生组织，与FLOWERING LOCUS D（FD）形成FT-FD复合物，从而促进植物开花[8]。TFL1是和FT序列相似、功能相反的植物开花调控基因[9]，通过抑制茎端分生组织形成花原基，延迟植物由营养生长向生殖生长的转变，在植物体内，通过FT和TFL1两个开花相关蛋白的平衡调控而诱导开花[10]。

FT基因在大多数植物体内具有保守的功能，FT超表达能促进水稻、烟草、野甘菊和龙胆草等短日照植物、拟南芥等长日照植物和番茄等中性植物提早开花，而且能使柑橘、毛果杨、苹果、猕猴桃、蓝莓和小桐子等多年生植物提早开花[11-12]。但是FT同源物同样存在抑制开花的情况[13⇓⇓⇓⇓-18]，如向日葵、甜菜、烟草、龙眼、大豆和甘蔗。

本课题组前期的研究发现，橡胶树中2个FT的同源基因HbFT1和HbFT2转化拟南芥后均有早花的表型，转化烟草和同为大戟科的小桐子也有早花的表型（结果未发表），对橡胶树一年中不同月份的表达分析初步确定HbFT2可能是橡胶树FT的候选基因[19]。因此，本研究将这2个基因的超表达载体均进行橡胶树的遗传转化，获得了阳性胚，对阳性胚进行编号，如正常胚则直接出苗，畸形胚则通过次生体胚发生进行再次体胚发生，将再次体胚发生获得的正常胚进行植株再生，并通过芽接的方式对阳性植株扩繁转基因阳性植株，通过对其进行表型观察、转基因验证及目的基因表达分析，对橡胶树开花调控基因FT的功能进一步分析，并为橡胶树转基因研究提供实验材料。

1 材料与方法

1.1 材料

本研究使用的转基因外植体材料为热研87- 6-62的体细胞胚，2014年通过转化体细胞胚的方法[20]侵染体细胞胚，分别获得HbFT1阳性胚状体2个，HbFT2阳性胚状体3个，并通过GUS染色鉴定为阳性，当代再生植株或经次生体胚发生扩繁后再生的植株通过芽接的方式扩繁获得HbFT1和HbFT2的超表达转基因阳性植株分别有4株和7株，于2017年9月定植在中国热带农业科学院儋州五队隔离网室，具体的阳性植株情况见表1。

表1 HbFT1和HbFT2转化橡胶树热研87-6-62体细胞胚获得的阳性植株

Tab. 1 The positive plantlets of HbFT1 and HbFT2 transformed into somatic embryos of rubber tree Reyan 87-6-62

外植体
Explant 外源基因
Exogenous gene 阳性植株编号
No. of positive plantlet 热研87-6-62体细胞胚 HbFT1 Ft2014604-2 热研87-6-62体细胞胚 HbFT1 Ft2014604-3 热研87-6-62体细胞胚 HbFT1 Ft2014557-2 热研87-6-62体细胞胚 HbFT1 Ft2014557-3 热研87-6-62体细胞胚 HbFT2 2014D276-2 热研87-6-62体细胞胚 HbFT2 2014D276-3 热研87-6-62体细胞胚 HbFT2 2014D214-1 热研87-6-62体细胞胚 HbFT2 2014D214-3 热研87-6-62体细胞胚 HbFT2 2014D187-1 热研87-6-62体细胞胚 HbFT2 2014D187-2 热研87-6-62体细胞胚 HbFT2 2014D187-3

1.2 方法

1.2.1 阳性植株PCR鉴定

选择HbFT1和HbFT2转基因阳性植株淡绿期的叶片，DNA提取方法详见天根生化科技（北京）有限公司的DNA提取试剂盒（DP320），采用NanoDrop 2000分光光度计和凝胶电泳检测DNA样品的浓度和完整性。根据HbFT1和HbFT2的CDS区设计基因的特异引物（表2），设计GUS和Npt基因引物（表2），采用PCR鉴定阳性植株，PCR扩增的酶使用TOLOBIO的2×Magic Green Taq SuperMix（21502），PCR反应体系为：2 μL DNA模板，引物各0.3 μL（10 μmol/L），7.5 μL 2×Magic Green Taq SuperMix，4.9 μL ddH2O。PCR反应程序为：94 ℃预变性3 min；94 ℃变性30 s，引物58 ℃复性10 s，72 ℃延伸1 min，35个循环；72 ℃终延伸10 min；4 ℃保存。PCR产物采用1%的琼脂糖凝胶电泳检测。每次PCR反应过程中，分别以ddH2O做空白对照（CK），未经转化的橡胶树叶片基因组DNA为阴性对照（CK‒），含有目的基因的表达载体质粒为阳性对照（CK+）。

表2 HbFT1、HbFT2、GUS、Npt基因扩增引物

Tab. 2 The amplification primers of HbFT1, HbFT2, GUS and Npt genes

基因
Gene 引物名称
Primer name 序列（5ʹ‒3ʹ）
Sequence (5ʹ‒3ʹ) HbFT1 HbFT1-528-F1 ATGCCTAGTGACAGAGATCCTCTT HbFT1-528-R1 TTAGCATCTTCTTCCCCCGG HbFT2 HbFT2-528-F1 ATGCCTAGAGATCCTCTGGTTGT HbFT2-528-R1 TTATTGTCCTCGCCTTCTTCCCC GUS GUS-5M GGTGGGAAAGCGCGTTACAAG GUS-6M GTTTACGCGTTGCTTCCGCCA Npt Npt-F TCAGAAGAACTCGTCAAGAAG Npt-R ATCATGGCTGATGCAATGCG

1.2.2 阳性植株GUS染色分析

利用Biosharp的GUS染色剂试剂盒（BL622A）进行GUS染色。将配置好的X-gluc溶液和缓冲液按照比例混合配成GUS染色工作液，取每个单株的适量叶片置于1.5 mL的离心管中，加入配制好的GUS染色工作液，置于37 ℃培养箱过夜，弃染色液，并加入浓度为75%的酒精洗脱叶片的颜色，直至变成白色或者浅黄色，拍照观察。

1.2.3 qRT-PCR分析

对上述转基因阳性植株进行qRT-PCR验证，分析促进开花和抑制开花基因在不同阳性植株的表达模式。根据橡胶树早花基因HbFT1、HbFT2和抑制开花基因HbTFL1、HbTFL2、HbTFL3、HbCEN1、HbCEN2的基因组序列，设计特异RT-qPCR引物（表3）。利用天根生化科技（北京）有限公司的RNAprep Pure多糖多酚植物总RNA提取试剂盒（DP441）提取样品总RNA。采用NanoDrop 2000分光光度计和凝胶电泳检测RNA样品的浓度和完整性。利用全式金反转录试剂盒（TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix）反转录成cDNA。将上述反转录得到的cDNA作为模板，以HbYLS8为内参基因，利用2×Q3 SYBR qPCR Master Mix（Universal）（22204，TOLOBIO）进行qPCR，反应体系与扩增程序按照说明书进行，实验设置3个技术重复。采用2‒ΔΔCt法计算基因的相对表达水平。

表3 qRT-PCR引物

Tab. 3 Primers for qRT-PCR

引物名称
Primers name 序列（5ʹ‒3ʹ）
Sequence (5ʹ‒3ʹ) HbYLS8-F CCTCGTCGTCATCCGATTC HbYLS8-R CAGGCACCTCAGTGATGTC HbFT1-F TACACATCAACGGTACACACCAGTC HbFT1-R CCAAAACATCCCCTATCACACG HbFT2-F ATCTTTCTACCATTTGACCCCATA HbFT2-R ATAACTCGCCCGCTTGTAATCT HbTFL1-F CCTCCTTAATCCCTGGCTACG HbTFL1-R ACATCGCCTACAACTCTCCCT HbTFL2-F GCCACATTTGGAAGGGAGATAG HbTFL2-R TAACACTTATTATTGAAGGCCACTTG HbTFL3-F ATACACCTCTTAGCCAGACGCTC HbTFL3-R TGTCGCCTCCTTGGACCTCA HbCEN1-F CTAATGGCGAAGACAACAGAT HbCEN1-R CACCTCCCTGGACCTCAAC HbCEN2-F GAAACAGCAGCAAGGAGACG HbCEN2-R ATTGGTGCGACTGATACGAC

2 结果与分析

2.1 转HbFT1和HbFT2阳性植株的PCR鉴定

利用3对PCR特异引物对上述的转基因植株进行扩增，结果如图1所示，在空白对照（CK）、阴性对照（CK‒）中均未扩增出相应片段大小的产物，而在阳性对照（CK+）和全部阳性单株中均能扩增出符合目标基因的片段大小，带型清晰。结果表明，所有的HbFT1和HbFT2阳性植株检测结果均为阳性。

图1 3对PCR特异引物对转基因植株的扩增结果

M：2K Plus DNA marker；A、B、C中4~7为转基因阳性植株Ft2014604-2、Ft2014604-3、Ft2014557-2、Ft2014557-3；

D、E、F中4~10为转基因阳性植株2014D276-2、2014D276-3、2014D214-1、2014D214-3、2014D187-1、2014D187-2、2014D187-3。

Fig. 1 PCR amplification results of transgenic plants by three pairs of PCR specific primers

M: 2K Plus DNA marker; 4-7 is the transgenic positive plants of Ft2014604-2, Ft2014604-3, Ft2014557-2 and Ft2014557-3 in picture A, B and C; 4-10 is transgenic positive plants 2014D276-2, 2014D276-3, 2014D214-1, 2014D214-3, 2014D187-1, 2014D187-2 and 2014D187-3 in picture D, E and F.

Full size|PPT slide

2.2 转HbFT1和HbFT2阳性植株的GUS染色分析

叶片GUS染色过夜并脱色后的结果如图2所示，GUS染色在叶片的边缘处居多，并且全部叶片边缘处都有深蓝色，而非转基因植株的叶片无染色。结果表明，GUS基因在这些叶片中均有表达，这与PCR检测的结果一致，证明这些转化植株确实为阳性植株。

图2 不同转基因单株的GUS染色结果

Fig. 2 The GUS staining of different positive plantlets

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2.3 HbFT1和HbFT2在超表达阳性植株中的qRT-PCR分析

为进一步解析超表达橡胶树中HbFT1和HbFT2的基因表达情况，对橡胶树HbFT1和HbFT2的超表达植株进行qRT-PCR分析，结果如图3所示，HbFT1基因在超表达植株（Ft2014604-2、Ft2014604-3、Ft2014557-2、Ft2014557-3）的表达量均显著高于野生型对照热研87-6-62，且Ft2014557-2和Ft2014557-3不同芽接树间的表达量也差异显著；而HbFT2基因在超表达植株2014D187-2、2014D276-2中的表达量显著高于野生型对照热研87-6-62，其他超表达植株与野生型的表达相当或者不及野生型的表达，不同芽接树间的表达差异较大，尤其是在2014D276胚和2014D187胚之间的差异达到显著水平。目前转基因阳性植株均已定植7 a，但获得的所有提早开花基因超表达植株均未提前开花。

图3 HbFT1和HbFT2基因在超表达植株中的表达分析

不同小写字母表示处理间差异显著（P<0.05）。

Fig. 3 The qRT-PCR analysis of HbFT1 and HbFT2 in over-expressed plantlets

Different lowercase letters indicate significant difference among treatments (P<0.05).

Full size|PPT slide

2.4 抑制开花基因在超表达阳性植株中的qRT- PCR分析

橡胶树中也存在抑制开花基因HbTFL1、HbTFL2、HbTFL3、HbCEN1和HbCEN2[21]，为更好地解析提早开花超表达植株未得到早花表型的原因，本研究同时在这些植株中进行抑制开花基因的表达分析。选择HbFT1和HbFT2超表达植株进行抑制开花基因的表达分析，结果如图4所示，在HbFT1超表达植株中，抑制开花的基因的表达绝大多数高于促进开花基因的表达，其中在Ft2014557-2中，抑制开花基因HbCEN2和HbTFL1的表达较高，均高于野生型及HbFT1的表达。而芽接扩繁的另一株Ft2014557-3中，HbTFL1和HbTFL2的表达较高，也均高于野生型和HbFT1的表达，但两株间表达有所差异。在Ft2014604-2和Ft2014604-3中，抑制开花基因HbCEN2、HbTFL1、HbTFL2的表达均高于野生型及HbFT1的表达。在HbFT2超表达植株中，抑制开花的基因的表达同样显著高于促进开花的基因表达（图5），表达不尽相同。在2014D214-1中，抑制开花的基因HbCEN2、HbTFL1和HbTFL2显著高于野生型和HbFT2的表达；而在芽接扩繁的另一株2014D214-3中， HbCEN2和HbTFL1的表达显著高于野生型和HbFT2的表达，但HbTFL2的表达与野生型无差异。在2014D276-2中，除HbCEN1的表达没有野生型高之外，其他抑制开花的基因的表达均显著高于野生型和HbFT2基因的表达；而在芽接扩繁的另一株2014D276-3中，只有HbTFL1、HbTFL2和HbTFL3显著高于野生型和HbFT2基因的表达。在2014D187-1中，HbTFL1和HbTFL2的表达显著高于野生型和HbFT2基因的表达；而在芽接扩繁的另外两株2014D187-2和2014D187-3中，只有HbTFL1的基因表达显著高于野生型和HbFT2基因的表达。

图4 HbFT1转基因阳性植株中不同开花基因的表达

不同小写字母表示差异显著（P<0.05）。

Fig. 4 The qRT-PCR analysis of flowering genes in over-expressed HbFT1 plantlets

Different lowercase letters indicate significant difference (P<0.05).

Full size|PPT slide

图5 HbFT2转基因阳性植株中不同开花基因的表达

不同小写字母表示差异显著（P<0.05）。

Fig. 5 The qRT-PCR analysis of flowering genes in over-expressed HbFT2 plantlets

Different lowercase letters indicate significant difference (P<0.05).

Full size|PPT slide

3 讨论

3.1 橡胶树体胚遗传转化的嵌合型导致芽接树之间的表达量差异较大

体细胞胚胎发生是一个能有效减少嵌合体植物产生的再生体系，因为诱导产生胚胎的是单个细胞[22]。在橡胶树的次生体胚发生中体细胞胚形成的石蜡切片观察研究发现，橡胶树次生体胚属于直接体胚发生途径，具有以体胚子叶表皮层中的胚性原始细胞的单细胞起源及子叶内部的多细胞起源2种起源方式[23-24]，而且显微镜下瞬时GUS表达分析证实，表皮细胞和表皮下细胞均存在农杆菌的侵染[20]。理论上讲，单细胞起源方式的体细胞胚胎发生应该可以得到遗传稳定的转基因植株，并且还可以有效地避免嵌合体的出现，但是橡胶树次生体胚的产生是单细胞起源和多细胞起源2种方式均存在。本研究获得的不同芽接树之间基因的表达量差异较大，而不同芽接树均来自于同一个阳性体细胞胚。HbFT2的表达结果显示，7株转基因材料中仅有2株HbFT2的表达高于野生型，其他5株均与野生型持平，而与这2株分别来自同一个阳性胚的另外的芽接苗与其表达差异大。HbFT1的表达结果也类似，虽然2个株系的2个植株的表达均高于野生型，但是其中1个阳性胚芽接产生的2个单株间的表达也呈现显著的差异。因此推测造成株系间表达差异大的原因是和阳性体细胞胚为嵌合体有关，UDAYABHANU等[24]也提出嵌合现象需要在进一步的转基因实验中得到证实。关于转基因嵌合体现象，一般有以下几种解释机制，如转化细胞和未转化细胞混合产生的芽，转基因的短暂表达或转基因的丢失。嵌合体经常存在于初代转基因植物中，对于杨树等多年生木本乔木，获得纯合子也相对比较困难，也需要经过二次再生获得较高频率的纯合子[25]。而在最新的橡胶树基因编辑的研究中也发现，超过90%的第0代转化胚胎是嵌合的，因此，从T0阳性胚胎中再生转基因植株不合适。在T1代纯合子胚的比例显著提高到接近50%[26]。针对以橡胶树体细胞胚为外植体获得初代阳性胚存在嵌合体的问题，笔者认为可以通过3个方面加以改进：一是继续将初代阳性胚次生体胚发生增殖到第二代或者第三代，再进行出胚及出苗；二是在转基因筛选过程中，选择可视化或者荧光显微镜可以很好地观察阳性胚的报告基因，如RUBY、DsRed或RFP等进行实时跟踪，将有指示标记的胚块或者胚进行后续继代实验；三是进一步研究橡胶树体细胞胚次生体胚发生过程中的细胞起源模式，从源头上避免嵌合体的产生。这些措施可以在将来的转基因过程中减少嵌合体的产生。

3.2 拮抗基因的平衡调控决定表型性状

拮抗基因是一对序列相似但功能相反的调控基因，最早承认SFT/SP影响植株形态和产量是从番茄SP基因对于表型的影响[27-28]。如PARK等[29]利用人工EMS诱变sp番茄，获得弱功能的SP和SFT突变基因，然后使杂合的SP和SFT基因达到一定的平衡状态，提高花序密度的同时，提高了营养生长能力，植株变得更加茂盛，最终提高了番茄单株的产量；棉花的GhSFT和GhSP基因产物的有目的的操作为增强有限花序的形状和在区域特异性环境下改变棉花的农耕带来希望[30]。在大豆中，功能性拮抗基因GmFT4和GmFT2a/5a的平衡决定了大豆的开花时间[17]，FT和TFL1通过竞争共同的下游FD与14-3-3等蛋白形成的复合体实现植物开花控制[31⇓⇓-34]。FT/TFL1平衡关系或剂量效应与植株形态建成具有显著相关性，即FT/TFL1表达比值决定了开花时间的长短与形态建成[12,35]。一般橡胶树在定植3~4 a即可开花，而本研究中转化早花基因在定植7 a也未得到早花表型，推测可能是由于橡胶树抑制开花基因的高表达所致。qRT-PCR分析结果显示HbTFL1在所有超表达促进开花基因的植株中均显著高于野生型与对应的超表达基因，推测该基因可能与抑制开花相关，因超表达促进开花的基因后，打破了FT/TFL1的平衡关系，抑制开花的基因表达也上调，而且超过促进开花基因的表达，最终导致超表达促进开花基因的橡胶树并未提前开花。本研究认为，在橡胶树这种基因组复杂的物种中，多拷贝基因经常存在，橡胶树转基因研究需要获得较大群体观察表型才具有可靠性，另外通过对拮抗基因的RNAi来改变剂量效应，以期获得需要的表型。

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摘要

在合适的时间开花对大多数植物的生存和成功繁衍极为重要。开花时间受错综复杂的环境因素和植物自身的遗传因子影响, 由开花调控因子所构成的光周期、春化、温度、赤霉素、自主以及年龄等至少6条既相互独立又相互联系的遗传途径调控。该文综述了有关拟南芥(Arabidopsis thaliana)开花时间调控的分子机制的最新研究进展, 并对今后的研究进行了展望。

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