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Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (9): 1327-1337.doi: 10.3724/SP.J.1006.2019.82069

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2019-09-12 Published:2019-05-17
  • Contact: Cai-Li BI E-mail:beicaili@sina.com
  • 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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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

Fig. 1

PCR amplification of OsUBA gene in rice M: 5 kb DNA marker; 1: PCR product."

Fig. 2

Conserved domain prediction of OsUBA protein"

Fig. 3

Phylogenetic tree of OsUBA and its homologues in plants The star indicates OsUBA."

Fig. 4

PCR amplification of OsUBA promoter M: 5 kb DNA marker; 1: PCR product."

Table 1

Prediction of cis-elements of OsUBA promoter by PlantCARE software"

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)

Fig. 5

Identification of OsUBA p:Gus vector by double digestion M: 5 kb DNA marker; 1: plasmid of pCAMBIA1391-OsUBAp (undigested); 2: identification of pCAMBIA1391-OsUBAp plasmid by Hind III/Sal I digestion."

Fig. 6

Analysis of Gus expression in OsUBA p:Gus transgenic rice plants WT: rice cv. Nipponbare; L6 and L2: two homozygous rice lines harboring OsUBA p:Gus."

Fig. 7

Expression of OsUBA in different organs and tissues A: roots and leaf from rice seedlings grown in normal conditions for 7 d; B: the main stem node during flowering; C: the 2nd internode of the main stem during flowering; D: flower; E: seed germinated for 1 d; F: seed germinated for 3 d. The scale in A represents 1 cm, and the scale in B and C equals to 2 mm, the scale in D, E, and F means 1 mm."

Fig. 8

Expression of OsUBA under ABA and GA treatments A: untreated control, normally grown for 24 h; B: 200 μmol L-1 ABA treatment for 24 h; C: 100 μmoL L-1 GA treatment for 24 h; Bar = 1 mm."

Fig. 9

Identification of 35S:OsUBA vector by PCR and double digestion A: amplification of positive clone harboring pCAMBIA1300- OsUBA; M: 5 kb marker; 1: positive clone; B: identification of pCAMBIA1300-OsUBA positive clone by Xba I/Kpn I digestion; M: 5 kb marker; 1: plasmid of pCAMBIA1300-OsUBA (undigested); 2: Xba I/Kpn I digestion of pCAMBIA1300-OsUBA plasmid."

Fig. 10

Relative expression analysis of OsUBA in OsUBA- overexpressing rice plants"

Fig. 11

Comparison of flowering time between OsUBA-overexpressing rice plants and the wild type control"

Fig. 12

ABA and GA treatment affects seed germination of OsUBA-overexpressing rice plants and the wild type control A: The seed germination course of the OsUBA-overexpressing rice plants and the wild type control on 1/2 MS medium; B: 1/2 MS medium supplemented with 100 μmol L-1 GA; C: 1/2 MS medium supplemented with 3 μmol L-1 ABA."

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