遗传 ›› 2023, Vol. 45 ›› Issue (8): 700-714.doi: 10.16288/j.yczz.22-392
• 研究报告 • 上一篇
陈凯(
), 王灏, 陈燚婷, 符可, 韩之刚, 李聪, 斯金平, 陈东红()
收稿日期:2023-02-28
修回日期:2023-07-13
出版日期:2023-08-20
发布日期:2023-07-24
通讯作者:
陈东红
E-mail:13588276311@qq.com;chendh212@163.com
作者简介:陈凯，硕士，研究方向：药用植物遗传育种。E-mail: 基金资助:
Kai Chen(), Hao Wang, Yiting Chen, Ke Fu, Zhigang Han, Cong Li, Jinping Si, Donghong Chen()
Received:2023-02-28
Revised:2023-07-13
Online:2023-08-20
Published:2023-07-24
Contact:
Donghong Chen
E-mail:13588276311@qq.com;chendh212@163.com
Supported by:摘要：
WUSCHEL-Related Homeobox (WOX)家族是植物特有的一类转录因子，在干细胞维持和器官形态建成等发育过程中发挥重要的调控作用。兰科珍稀名贵中药植物铁皮石斛(Dendrobium catenatum)具有独特的附生生活方式和生长发育特性，对其WOX家族基因功能探究有助于进一步了解铁皮石斛发育的保守性和特异性。本研究对铁皮石斛WOX家族基因(DcWOX)进行了系统进化、组织表达模式、异源表达功能检测等分析。结果显示，铁皮石斛WOX家族基因可分为3个进化支，具有显著差异的组织表达谱；在转基因拟南芥中，DcWOX4过表达导致植株显著矮小，叶缘羽状深裂，花期推迟2周；DcWOX9过表达导致植株矮化，叶缘锯齿状，开花推迟1周，强表型植株雌雄蕊均不育；DcWOX11过表达导致叶缘向下卷曲；DcWOX4/9/11过表达拟南芥叶片形态建成异常与TCP家族基因和CUC家族基因的下调以及KNOX家族基因的上调有关；花期推迟与FT、SOC1和CO等早花基因的下调有关。因此，本研究表明铁皮石斛WOX家族基因在调控植株形态建成、叶发育、开花时间和育性等方面具有重要功能，进一步拓展了对WOX家族基因的功能了解，为深入探讨兰科植物进化与发育的保守性和独特性提供参考。
陈凯, 王灏, 陈燚婷, 符可, 韩之刚, 李聪, 斯金平, 陈东红. 铁皮石斛WOX家族基因在生长发育中的功能分析[J]. 遗传, 2023, 45(8): 700-714.
Kai Chen, Hao Wang, Yiting Chen, Ke Fu, Zhigang Han, Cong Li, Jinping Si, Donghong Chen. Functional analysis of WOX family genes in Dendrobium catenatum during growth and development[J]. Hereditas(Beijing), 2023, 45(8): 700-714.
表1
本研究所用引物信息"
| 基因 | 引物序列(5′→3′) | 用途 |
|---|---|---|
| DcWOX4 | F：ATGAAGTTTCAGCAGTTGGAAT | 基因克隆、半定量PCR |
| R：AAAGTAGTTATTTCCATTAATGTTC | ||
| DcWOX9 | F：ATGGCTTCATCAAACAGGCAC | 基因克隆、半定量PCR |
| R：CTGAACCAAATAATAGGATGCTC | ||
| DcWOX11 | F：ATGGTAGCTATAAATACTTCCAA | 基因克隆 |
| R：ATTAGGCCTAGCAATCAAGTA | ||
| DcWOX3B | F：ATGCCACAGGTTCCATCAACAAG | 基因克隆、半定量PCR |
| R：GTTGGTTGAAGCCGAGCATGAG | ||
| DcWOX2 | F：CAACAGGCATAATGGAGA | 半定量PCR |
| R：CACAACCTTCTTCATCCT | ||
| DcWOX3A | F：CATAAGGCAAGAGATAGACA | 半定量PCR |
| R：GAGGAAGAGAAGCAGAAG | ||
| DcWOX10 | F：GCCATGTCACTGATGTTAA | 半定量PCR |
| R：TCAATCTCTGCCTCACTT | ||
| DcWOX11 | F：ATCATCATCTTCTTCTTCT | 半定量PCR |
| R：GAACCTCTGTAAGTATCC | ||
| DcWOX12 | F：ATGGAAGACAACCAAACCAAT | 半定量PCR |
| R：AGTAGGTCTTGCAACCAAGTAGT | ||
| DcWOX13 | F：GACTACTCATCATTCTTC | 半定量PCR |
| R：CTTCTGTTGTTATATCTGT | ||
| DcACT7 | F：GATTCTCAGGCTTGACCTTGC | 半定量PCR |
| R：CTGGACCAGATTCATCATATTCG | ||
| FT | F：CTTGGCAGGCAAACAGTGTATGCAC | qRT-PCR |
| R：GCCACTCTCCCTCTGACAATTGTAGA | ||
| FLC | F：CTAGCCAGATGGAGAATAATCATCATG | qRT-PCR |
| R：TTAAGGTGGCTAATTAAGTAGTGGGAG | ||
| CO | F：TCAGGGACTCACTACAACGACAATGG | qRT-PCR |
| R：TTGGGTGTGAAGCTGTTGTGACACAT | ||
| SOC1 | F：AGCTGCAGAAAACGAGAAGCTCTCTG | qRT-PCR |
| R：GGGCTACTCTCTTCATCACCTCTTCC | ||
| MAF4 | F：TGGCCAAGATCCTCAGTCGTTATGA | qRT-PCR |
| R：GCTGCTCTTCCAGGGACTTTAGACA | ||
| MAF5 | F：GATGGAGCTTGTGAAGAACCTTCAGG | qRT-PCR |
| R：CAGCCGTTGATGATTGGTGGTTACTTG | ||
| STM | F：GCAACACATCCTCACCATTACTTCA | qRT-PCR |
| R：ATCAAAGCATGGTGGAGGAGA | ||
| BP | F：TCCCATTCACATCCTCAACA | qRT-PCR |
| R：CCCCTCCGCTGTTATTCTCT | ||
| KNAT2 | F：AAACGCCATTGGAAGCCT | qRT-PCR |
| R：ACAATGCACAATTTCATGTCTCTCT | ||
| KNAT6 | F：CCAAGAGAAGCAAGACAAGCTC | qRT-PCR |
| R：CAGCTAATGCTATCTTATCTCCTTCAG | ||
| AtActin | F：TGGCATCAYACTTTCTACAA | qRT-PCR |
| R：CCACCACTDAGCACAATGTT |
图1
WOX家族成员系统进化关系 Ao：芦笋(A. officinalis)；As：深圳拟兰(A. shenzhenica)；At：拟南芥(A. thaliana)；Dc：铁皮石斛(D. catenatum)；Dn：金钗石斛(D. nobile)；Dm：鼓槌石斛(D. chrysotoxum)；Pe：小兰屿蝴蝶兰(P. equestris)；Vp：香荚兰(V. planifolia)； Os：水稻(O. sativa)。DcWOX2：LOC110104455；DcWOX3A：LOC110110752；DcWOX3B：LOC110111262；DcWOX4：LOC110092438；DcWOX5：LOC110093208；DcWOX9：LOC110093317；DcWOX10：LOC110095151；DcWOX11：LOC110107133；DcWOX12：LOC110108675；DcWOX13：LOC110101510；PeWUS：LOC110036538；PeWOX2：LOC110038808；PeWOX3：LOC110026487；PeWOX4：LOC110026126；PeWOX5：LOC110022083；PeWOX9A：LOC110036348；PeWOX9B：LOC110034479；PeWOX10：LOC110033171；PeWOX11：LOC110026463；PeWOX12：LOC110019832；PeWOX13A：LOC110022094；PeWOX13B：LOC110019714；AsWUS：AXF42_Ash012361；AsWOX2A：AXF42_Ash017453；AsWOX2B：AXF42_Ash017709；AsWOX3A：AXF42_Ash020593；AsWOX3B：AXF42_Ash020021；AsWOX7：AXF42_Ash019125；AsWOX9：AXF42_Ash018680；AsWOX11：AXF42_Ash008663；AsWOX12：AXF42_Ash008813；AsWOX13：AXF42_Ash009782；AoWUS：LOC109849707；AoWOX2：LOC109821937；AoWOX3：LOC109849426；AoWOX5：LOC109849790；AoWOX9：LOC109842428；AoWOX10：LOC109837258；AoWOX13：LOC109820982；DmWOX2：KAH0458396.1；DmWUS：KAH0470781.1；DmWOX3A：KAH0470737.1；DmWOX3B：KAH0470646.1；DmWOX5：KAH0454885.1；DmWOX4：KAH0461191.1；DmWOX13：KAH0463007.1；DmWOX10：KAH0449253.1；DmWOX12：KAH0465957.1；DmWOX11：KAH0454260.1；VpWOX2：KAG0463759.1；VpWOX4：KAG0479964.1；VpWOX3：KAG0460903.1；VpWUSA：KAG0457289.1；VpWUSB：KAG0453191.1；VpWOX9：KAG0458137.1；VpWOX13：KAG0488299.1；VpWOX11：KAG0477525.1；DnWOX2：KAI0502012.1；DnWUS：KAI0530632.1；DnWOX3A：KAI0531433.1；DnWOX4：KAI0512419.1；DnWOX3B：KAI0498241.1；DnWOX5：KAI0498434.1；DnWOX9：KAI0513400.1；DnWOX13：KAI0514107.1；DnWOX10：KAI0489033.1；DnWOX12：KAI0520058.1。"
图2
DcWOX基因组织表达谱 A：DcWOX基因半定量PCR电泳图；B：DcWOX基因表达热图。CK：空白对照；Ro：根；St：茎；Le：叶；Fb：花蕾；Fl：花；Fr：果；Pr：原球茎；Po：花粉块；Gy：雌蕊。"
图3
DcWOX4-GFP转基因拟南芥植株的表型观察与转录组分析 A：四周龄拟南芥；B：四周龄莲座叶；C：角果；D：六周龄成株；E：转基因拟南芥鉴定(PCR鉴定和根尖GFP荧光观察)；F：3个转化系植株T3代最大莲座叶直径、六周龄植株高度、开花时间、角果长度统计；G：两周龄拟南芥转录组GO富集分析；H：两周龄拟南芥调控叶发育和开花时间的差异表达基因；I：四周龄拟南芥叶发育调控基因qRT-PCR；J：四周龄拟南芥开花时间调控基因qRT-PCR。比例尺A~D为1 cm，E为50 μm。图表数据显著性分析采用Student t-test，*P<0.01，**P<0.001。"
图4
DcWOX9-GFP转基因拟南芥植株表型的表型观察与转录组分析 A：四周龄WOX9-GFP过表达拟南芥；B：四周龄莲座叶；C：花；D：角果；E：六周龄成株；F：转基因拟南芥鉴定(植株RNA提取PCR鉴定和根尖GFP荧光)；G：转化系植株T3代(共12株)最大莲座叶直径、六周龄植株高度、开花时间、角果长度统计；H：两周龄拟南芥转录组GO富集分析；I：两周龄拟南芥调控叶发育及开花时间的差异表达基因；J：四周龄拟南芥叶发育调控基因qRT-PCR；K：四周龄拟南芥开花时间调控基因qRT-PCR。比例尺：A、B、D、E为1 cm，C为0.05 cm，F为50 μm。图表数据显著性分析采用Student t-test，*P<0.01，**P<0.001。"
图5
DcWOX9-GFP转基因拟南芥植株强表型的叶形态发育形态观察与花器官形态观察 A~D：分别为4、5、6、8周DcWOX9转基因拟南芥强表型植株；E：花序；F：花形态；G：雄蕊亚历山大染色；H~K：两个转化系T1代共两株的花器官数量、莲座叶直径、开花时间、植株最大高度统计。比例尺：A~D为1 cm，E为0.5 cm，F为0.05 cm，G为0.01 cm。图表数据显著性分析采用Student t-test，*P<0.01，**P<0.001。"
图6
DcWOX11-GFP转基因拟南芥植株的表型观察与转录组分析 A：四周龄拟南芥；B：四周龄莲座叶；C：角果；D：六周龄成株；E：转基因拟南芥鉴定(植株RNA提取PCR鉴定和根尖GFP荧光)；F：3个转化系植株T3代最大莲座叶直径、六周龄植株高度、开花时间、角果长度统计；G、H：两周龄拟南芥转录组GO富集分析和叶发育及花时差异表达显著基因；I：四周龄拟南芥叶发育调控基因qRT-PCR；J：四周龄拟南芥开花时间调控基因qRT-PCR。比例尺为A~D为1 cm，E为50 μm。图表数据显著性分析采用Student t-test，*P<0.01，**P<0.001。"
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