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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (5): 1351-1360.doi: 10.3724/SP.J.1006.2024.32027

• RESEARCH NOTES • Previous Articles    

Mechanism of loding residence and drought tolerance of OsCNGC10 gene in rice

ZHU Zhong-Lin1(), WEN Yue1, ZHOU Qi1, WU Yan-Fei1, DU Xue-Zhu1,2,*(), SHENG Feng1,*()   

  1. 1School of Life Sciences, Hubei University / State Key Laboratory of Biocatalysis and Enzyme Engineering, Wuhan 430062, Hubei, China
    2Hubei Hongshan Laboratory, Wuhan 430062, Hubei, China
  • Received:2023-07-20 Accepted:2024-01-12 Online:2024-05-12 Published:2024-02-19
  • Contact: E-mail: shengfsk@163.com; E-mail: duxeuzhusk@163.com
  • Supported by:
    Wuhan Municipal Key Technology Project of Biotechnology(2022021302024851);Hubei Provincial Key Laboratory of Grain Crop Germplasm Innovation and Genetic Improvement(2018lzjj01)

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Abstract:

Cyclic nucleotide-gated ion channels are ligand-gated cationic channels that exist in animals and plants, which are an important part of eukaryotic signaling cascades. In this study, OsCNGC10 (cyclic nucleotide-gated channel) gene in rice was used, and the overexpression vector pU1301-CNGC10-Flag and the double-target knockout vector pRGEB32-CRISPR/cas9-cngc10 were constructed. The knockout and overexpression materials were obtained by Agrobacterium-mediated genetic transformation. Homozygous plants oscngc10-2 and OE-CNGC10-6 were isolated from T2 generation. The analysis of stem characteristics and lodging resistance of transgenic plants showed that oscngc10-2 had enhanced stem strength and lodging resistance. Stem cell wall sections and tissue composition analysis showed that oscngc10-2 increased lodging resistance due to the increase of stem wall thickness, parenchyma cell abundance, and lignin content. The knockout of OsCNGC10 increased the lignin content and the abundance of stem-cell wall parenchyma cells. The overexpression of OsCNGC10 reduced stem wall thickness, lignin content, and cell abundance in stem cell wall, while the knockdown of OsCNGC10 increased lignin content and increased the abundance of thin-walled cells in stem cell wall, suggesting that OsCNGC10 was associated with the composition of stem cell wall and negatively regulated lodging resistance in rice. T2 generation field experiment indicated that compared with the wild type, oscngc10-2 significantly increased plant height, the effective panicle length, panicle number, seed setting rate, 1000-grain weight, and yield per plant. The results of drought stress at seedling stage showed that malondialdehyde (MDA) content accumulated rapidly in OsCNGC10 defective plants under drought stress and insufficient free proline (Pro) was formed, while the free Pro content in OsCNGC10 plants was significantly increased. Moreover, the MDA accumulation rate was relatively slow, which preliminarily indicated that OsCNGC10 positively regulated the drought resistance at seedling stage. The results of this study indicated that OsCNGC10 might have a potential function in lodging resistance and drought resistance in rice, which providing a theoretical basis and new germplasm resources for the breeding lodging resistance and high yield of new rice varieties.

Key words: rice, OsCNGC10, lodging resistance, drought tolerance, cell wall

Table 1

Nucleotide sequence and corresponding primer sequences used in this study"

名称Primer name 序列Primer sequence (5'-3') 用途Usage
OsCNGC10-F ATGTTTGGGGCGGGGAAGGTGGACG 基因扩增
OsCNGC10-R TTACTCACAGGGTTCAGCTGAAAAAT Gene amplification
OsCNGC10-Flag-F GAACGATAGCCGGTACCATGTTTGGGGCGGGGAAGGT 带载体接头的基因扩增引物
OsCNGC10-Flag-R CTTTGTAATCGGATCCCTCACAGGGTTCAGCTGAAA Primers for gene amplification with vector connectors
L5AD5-F CAGATGATCCGTGGCAACaaagcaccagtggtctag 获得串联结构
L5AD5-R TTTCTAGCTCTAAAACaaaaaaaaaagcaccgactcg Obtaining a tandem structure
OsCNGC10-sgRNA1-F TCAAGAGGCAGAGAACCGTGgttttagagctagaaata 获得串联结构
OsCNGC10-sgRNA1-R CACGGTTCTCTGCCTCTTGAtgcaccagccgggaat Obtaining a tandem structure
OsCNGC10-sgRNA2-F CACGGTTCTCTGCCTCTTGAtgcaccagccgggaat 获得串联结构
OsCNGC10-sgRNA2-R CGCAATTTCCTTTGGATCCGtgcaccagccgggaat Obtaining a tandem structure
S5AD5-F CAGATGATCCGTGGCAACaaag 获得串联结构
S5AD5-R TTTCTAGCTCTAAAACaaaa Obtaining a tandem structure
OsCNGC10-target1-F GCGGTGTGGTTGACGAGTTC 靶点1序列
OsCNGC10-target1-R GCCAAATCACTCGCAGGTCG Sequence of target site 1
OsCNGC10-target2-F ATTGGGACAGACAGGCATTT 靶点2序列
OsCNGC10-target2-R GTCCTTAGTGTGGTCTGGGC Sequence of target site 2
Hyg-F ACGGTGTCGTCCATCACAGTTTGCC 转基因植株阳性鉴定
Hyg-R TTCCGGAAGTGCTTGACATTGGGG Identification of the positive transgenic plan
OsCNGC10-RT-F TACCACCACTGAGAACGATGT OsCNCG10表达量分析
OsCNGC10-RT-R TACCACCACTGAGAACGATGT Relative expression analysis of OsCNGC10

Fig. 1

PCR assay of OsCNGC10 overexpression vector colonies M: Trans 2K; 1-8: the colony PCR fragments."

Fig. 2

PCR assay of OsCNGC10 knockout terminal vector colonies M: Trans 2K; 1-8: the colony PCR fragments."

Fig. 3

Positive identification of transgenic plants in T0 generation (A): positive identification of transgenic plants; (B): the expression analysis of the overexpression lines; (C): target decoding analysis in knockout lines. M: Trans 2K; N: the negative control."

Fig. 4

Transgenic plants and wild type (WT) plant types (A): transgenic materials and field phenotypes of wild type at heading stage; (B): transgenic materials and wild type phenotypes at field maturity stage; (C): transgenic materials and single plant phenotypes of wild type at heading stages; (D): transgenic materials and single plant phenotypes of wild type at maturity stage."

Fig. 5

Basal internode morphology of transgenic plants and wild type (WT)"

Table 2

Strength analysis of transgenic plants and wild-type stems"

性状
Characteristics		 野生型日本晴
Wild type		 突变体
Mutant oscngc10-2		 超表达
Overexpression CNCG10-6
基部节间距Basal segmental spacing (cm) 9.51±1.36 4.88±0.88** 8.91±1.22
外茎 Outer stem (mm) 3.57±0.45 5.79±0.68* 3.25±0.12
内径 Inner diameter (mm) 2.34±0.45 3.89±0.68* 2.19±0.12
壁厚Thickness of wall (mm) 1.23±0.21 1.90±0.40* 1.24±0.13
茎秆强度Strength of stem (N) 8.13±1.18 24.97±8.02** 9.08±3.04

Fig. 6

Observation of transgenic plants and wild-type (WT) tissue sections CWT: cell wall thickness; SWT: straw wall thickness."

Fig. 7

Analysis of main components of cell wall of transgenic plants and wild type (WT) *: the correlation is significant at the 0.05 probability level. "

Table 3

Agronomic traits of transgenic plants and wild types"

性状
Characteristics		 野生型日本晴
Wild type		 突变体
Mutant oscngc10-2		 超表达
Overexpression CNCG10-6
生育期Growth period (d) 130 137 121
株高Plant height (cm) 73.23±2.91 90.97±3.22** 63.73±2.83
穗长Panicle length (cm) 15.16±1.43 18.35±2.25* 13.10±1.23
分蘖数Tiller number per plant 32.67±2.52 33.00±2.65 50.00±3.50**
每穗粒数Number of grain per panicle 105.00±4.99 237.00±8.85** 60.00±2.25
一次枝梗数Primary branch number per panicle 8.00±0.41 18.00±0.97* 8.00±0.39
二次枝梗数Secondary branch number per panicle 8.00±0.51 29.00±1.33* 3.00±0.12
千粒重Thousand-seed weight (g) 19.50±0.65 22.50±0.98* 15.10±0.56
理论产量Theoretical yield (t hm-2) 10.80±0.23 12.20±0.35* 6.90±0.11*
结实率Seed fertility (%) 86.50±0.68 93.80±1.90* 80.20±1.30

Fig. 8

Phenotype and survival rate of transgenic plants and wild type (WT) under drought stress for 15 days at seedling stage *: the correlation is significant at the 0.05 probability level. Bar: 20 cm."

Fig. 9

Proline and MDA contents of transgenic plants and wild type (WT) after 15 d drought stress at seedling stage A: proline content in the leaves of the plants; B: MDA content in the leaves of the plants. **: the correlation is significant at the 0.01 probability level; *: the correlation is significant at the 0.05 probability level. "

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