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作物学报 ›› 2019, Vol. 45 ›› Issue (9): 1327-1337.doi: 10.3724/SP.J.1006.2019.82069

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻OsUBA基因的表达及其在促进种子萌发和开花中的功能

张双双,王立伟,姚楠,郭光艳,夏玉凤,秘彩莉()   

  1. 河北师范大学生命科学学院, 河北石家庄 050024
  • 收稿日期:2018-12-25 接受日期:2019-05-12 出版日期:2019-09-12 网络出版日期:2019-05-17
  • 通讯作者: 秘彩莉
  • 作者简介:E-mail: zhangshuang1597@126.com
  • 基金资助:
    本研究由国家自然科学基金项目(31101129);河北师范大学博士基金资助(L2018B14)

Expression of OsUBA and its function in promoting seed germination and flowering

ZHANG Shuang-Shuang,WANG Li-Wei,YAO Nan,GUO Guang-Yan,XIA Yu-Feng,BI Cai-Li()   

  1. Life Science College, Hebei Normal University, Shijiazhuang 050024, Hebei, China
  • Received:2018-12-25 Accepted:2019-05-12 Published:2019-09-12 Published online:2019-05-17
  • Contact: Cai-Li BI
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31101129);the Doctoral Foundation of Hebei Normal University(L2018B14)

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摘要：

自噬是将功能异常或不需要的胞内组分降解的细胞学过程, 广泛参与真核生物的生长发育过程、对营养缺乏的响应及生物/非生物胁迫反应。NBR1 (Next to BRCA1 gene 1, NBR1)是在植物中发现的最重要的自噬受体, 但有关植物NBR1类自噬受体的研究较少, 水稻中此类蛋白的研究还是空白。本文通过RT-PCR方法, 从水稻日本晴幼苗的 cDNA中克隆到一个含有泛素相关结构域(Ubiquitin associated, UBA)的基因,将其命名为OsUBA。OsUBA的开放阅读框长2538 bp, 编码845个氨基酸残基。OsUBA属于水稻中的NBR1类蛋白。OsUBA的启动子区有多个与光、逆境胁迫及激素反应相关的元件; OsUBA基因在水稻花药、正在萌发的种子以及根中的表达量较高,在茎和叶中也有表达; 200 μmol L -1ABA处理显著抑制OsUBA的表达, 100 μmol L -1 GA处理后OsUBA的表达略有升高。对OsUBA过表达水稻株系的研究表明, 转基因水稻种子的萌发比野生型更快, ABA (3 μmol L -1)处理显著抑制OsUBA过表达水稻株系种子的萌发, GA (100 μmol L -1)处理对OsUBA过表达水稻株系种子的萌发略有促进; OsUBA过表达水稻株系的开花时间较野生型明显提前。这些结果表明, 水稻NBR1蛋白基因OsUBA的表达和功能可能与对开花时间和种子萌发的调控以及生物/非生物胁迫反应有关。

关键词: 水稻, NBR1, 转基因, 开花时间, 种子萌发

Abstract:

Autophagy is a cellular process by which dysfunctional or unnecessary cellular components are degraded. Autophagy plays important roles in growth and development, and makes responses to nutritional deficiency and biotic/abiotic stress responsiveness in eukaryotes. NBR1 (Next to BRCA1 gene 1, NBR1) proteins were the most important autophagy cargo receptors found in plants. Up to date, only limited reports about NBR1 were available in plants, and the function of NBR1 proteins in rice remains unclear. An UBA (Ubiquitin associated, UBA) domain-containing gene was cloned from cDNA of rice (Oryza sativa L. japonica cv. Nipponbare) seedlings and was named as OsUBA. The open reading frame of OsUBA is 2538 bp in length and encodes 845 amino acids residues. Conserved domain prediction and phylogenetic analysis suggested that OsUBA belongs to NBR1 proteins. Promoter analysis suggested that many motifs related to light-, stress-, and hormone responsiveness were predicted in OsUBA promoter sequence. Gus staining of the rice plants harboring OsUBA p:Gus showed that OsUBA was highly expressed in anthers, germinating seeds, and roots, OsUBA transcripts could also be detected in stems and leaves. Expression of OsUBA was dramatically inhibited by 200 μmol L -1 ABA treatment, and slightly increased by 100 μmol L -1 GA treatment. Compared with the wild type control, the OsUBA-overexpressors germinated more quickly and flowered obviously earlier. The seed germination of the OsUBA-overexpressed rice lines was obviously inhibited by 3 μmol L -1 ABA treatment, and promoted slightly by 100 μmol L -1GA treatment. These results suggested that the expression and function of OsUBA may be related to regulations of flowering time, seed germination and biotic/abiotic stress responsiveness in rice.

Key words: rice (Oryza sativa L.), NBR1, transgenic, flowering time, seed germination

图1

水稻OsUBA基因的PCR扩增 M: 5 kb DNA marker; 1: PCR 产物。"

图2

水稻OsUBA蛋白的保守结构域预测"

图3

OsUBA蛋白及其他植物中的同源蛋白的系统进化分析 星形所示为OsUBA。"

图4

OsUBA基因启动子的PCR扩增 M: 5 kb DNA marker; 1: PCR产物。"

表1

OsUBA基因启动子调控元件预测"

功能
Function		 元件名称(核心序列)
Element (core sequence)
ABA响应元件
cis-acting element involved in the abscisic acid responsiveness		 ABRE (ACGTG)
分生组织表达调控元件
cis-acting regulatory element related to meristem expression		 CAT-box (GCCACT)
茉莉酸甲酯响应元件
cis-acting regulatory element involved in the MeJA-responsiveness		 CGTCA-motif (CGTCA)
赤霉素响应元件
cis-acting element involved in gibberellin-responsiveness		 TATC-box (TATCCCA), P-box (CCTTTTG), GARE-motif (TCTGTTG)
水杨酸响应元件
cis-acting element involved in salicylic acid responsiveness		 TCA-element (CCATCTTTTT)
缺氧特异诱导增强子样元件
Enhancer-like element involved in anoxic specific inducibility		 GC-motif (CCCCCG)
功能
Function		 元件名称(核心序列)
Element (core sequence)
低温响应元件
cis-acting element involved in low-temperature responsiveness		 LTR (CCGAAA)
光响应相关元件
cis-acting regulatory element involved in light responsiveness		 G-box (CACGTC), Sp1 (CC(G/A)CCC)
部分光反应元件
Part of a light responsive element		 Box 4 (ATTAAT), GATA-motif (GATAGGG), I-box (GATAAGGTG), LAMP-element (CCTTATCCA), chs-CMA1a (TTACTTAA), chs-CMA2c (ATATACGTGAAGG)

图5

OsUBA p:Gus载体的酶切鉴定 M: 5 kb DNA marker; 1: pCAMBIA1391-OsUBAp质粒(未酶切); 2: pCAMBIA1391-OsUBAp质粒的Hind III/Sal I双酶切鉴定。"

图6

OsUBA p:Gus水稻株系中Gus的表达分析 WT: 野生型水稻日本晴; L6和L12: 2个OsUBA p:Gus水稻纯合株系。"

图7

OsUBA在不同器官和组织中的表达特性 A: 正常生长7 d水稻的根和叶; B: 开花期主茎的茎节; C: 开花期主茎的第2节间的横切面; D: 花; E: 萌发1 d的种子; F: 萌发3 d的种子。A图中的标尺代表1 cm; B图和C图中的标尺表示2 mm; D、E和F图中的标尺表示1 mm。"

图8

ABA和GA处理对OsUBA表达的影响 A: 未处理对照, 正常生长24 h; B: 200 μmol L-1 ABA处理24 h; C: 100 μmoL L-1 GA处理24 h; Bar = 1 mm。"

图9

35S:OsUBA载体的PCR及双酶切鉴定 A: pCAMBIA1300-OsUBA的菌液PCR; M: 5 kb marker; 1: 阳性克隆; B: pCAMBIA1300-OsUBA质粒的Xba I/Kpn I双酶切鉴定; M: 5 kb marker; 1: 阳性克隆质粒(未酶切); 2: 阳性克隆质粒的Xba I/Kpn I双酶切。"

图10

过量表达OsUBA的水稻株系中OsUBA的表达水平分析"

图11

OsUBA过表达水稻株系和野生型的开花时间比较"

图12

ABA和GA处理对OsUBA过表达水稻株系和野生型种子萌发的影响 A: 1/2 MS培养基上Osuba过表达的水稻株系种子萌发过程和野生型对照; B: 1/2 MS培养基添加100 μmol L-1 GA; C: 1/2 MS培养基添加3 μmol L-1 ABA。"

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