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作物学报 ›› 2024, Vol. 50 ›› Issue (5): 1351-1360.doi: 10.3724/SP.J.1006.2024.32027

• 研究简报 • 上一篇    

水稻OsCNGC10基因抗倒伏性以及抗旱性功能研究

朱忠林1(), 文月1, 周棋1, 巫燕飞1, 杜雪竹1,2,*(), 盛锋1,*()   

  1. 1湖北大学生命科学学院 / 省部共建生物催化与酶工程国家重点实验室, 湖北武汉 430062
    2湖北洪山实验室, 湖北武汉 430062
  • 收稿日期:2023-07-20 接受日期:2024-01-12 出版日期:2024-05-12 网络出版日期:2024-02-19
  • 通讯作者: 盛锋, E-mail: shengfsk@163.com; 杜雪竹, E-mail: duxeuzhusk@163.com
  • 作者简介:E-mail: 1418369969@qq.com
  • 基金资助:
    武汉市生物技术关键技术科技重大专项(2022021302024851);粮食作物种质创新与遗传改良湖北省重点实验室开放课题项目(2018lzjj01)

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 Published:2024-05-12 Published online: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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摘要：

环核苷酸门控离子通道是一种配体门控的阳离子通道, 存在于动物和植物体内, 是真核生物信号级联反应的重要组成部分。本研究利用水稻环核苷酸门控离子通道(cyclic nucleotide-gated channel, OsCNGC10)基因, 构建了超表达载体 pU1301-CNGC10-Flag和双靶点敲除载体pRGEB32-CRISPR/cas9-cngc10, 通过农杆菌介导的遗传转化法获得敲除和过表达材料, 并从T2代中分离到纯合植株oscngc10-2及OE-CNGC10-6。转基因植株茎秆特性以及抗倒伏性分析表明, 突变体oscngc10-2茎秆强度和抗倒伏性增强; 茎秆细胞壁组织切片以及组织成分分析则表明突变体oscngc10-2植株抗倒伏性增强是由于茎秆细胞壁茎壁厚度、薄壁组织细胞丰度以及木质素含量增加所致; 过表达OsCNGC10降低了茎秆壁厚、茎秆木质素含量以及茎秆细胞壁细胞丰度, 敲除OsCNGC10增加了茎秆木质素含量且增加了茎秆细胞壁薄壁细胞丰度, 初步证明OsCNGC10与水稻茎秆细胞壁成分合成相关, 负调控水稻抗倒伏性; T2代田间试验结果表明, 与野生型相比, 突变体oscngc10-2植株的株高、有效穗、穗长、穗粒数、结实率、千粒重和单株产量等农艺性状显著提升; 苗期干旱胁迫实验结果表明, 在干旱胁迫下, OsCNGC10基因缺陷型植株体内丙二醛(MDA)含量积累速度加快, 且无法形成足够的游离脯氨酸, 而过表达OsCNGC10植株在遭受干旱胁迫时, 体内游离脯氨酸(Pro)含量大量升高, 且MDA积累速度相对变慢, 初步说明OsCNGC10正向调控水稻苗期抗旱性。本研究结果表明水稻OsCNGC10可能在水稻抗倒伏及抗旱方面有潜在功能, 为培育抗倒伏且高产的水稻新品种提供了理论基础和新的种质资源。

关键词: 水稻, OsCNGC10, 抗倒伏, 抗旱, 细胞壁

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

表1

本研究所用核苷酸序列及相应引物"

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

图1

OsCNGC10超表达载体菌落PCR M: Trans 2K; 1~8: 菌落PCR片段。"

图2

OsCNGC10敲除终载体菌落PCR检测 M: Trans 2K; 1~8: 菌落PCR片段。"

图3

T0代转基因植株的阳性鉴定 (A): 转基因植株阳性鉴定; (B): 超表达株系表达量分析; (C): 敲除株系oscngc10-2靶点解码分析。M: Trans 2K; N: 阴性对照。"

图4

转基因植株及野生型(WT)株型 (A): 转基因材料以及野生型田间抽穗期表型; (B): 转基因材料以及野生型田间成熟期表型; (C): 转基因材料以及野生型抽穗期单株表型; (D): 转基因材料以及野生型成熟期单株表型。"

图 5

转基因植株及野生型(WT)基部节间形态"

表2

转基因植株与野生型茎秆强度分析"

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

图6

转基因植株与野生型(WT)组织切片显微镜观察 CWT: 茎壁厚度; SWT: 秆壁厚度。"

图7

转基因植株与野生型(WT)细胞壁主要成分分析 Cellulose: 纤维素; Lignin: 木质素; Hemicellulose: 半纤维素。 *表示在0.05概率水平显著相关。"

表3

转基因植株与野生型(WT)农艺性状分析"

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

图8

转基因植株与野生型(WT)苗期干旱胁迫15 d表型以及存活率 *表示在0.05概率水平显著相关。标尺为20 cm。"

图9

转基因植株与野生型(WT)苗期干旱胁迫15 d后Pro及MDA含量 A: 植株叶片脯氨酸含量; B: 植株叶片丙二醛含量。***表示在0.01概率水平显著相关; *表示在0.05概率水平显著相关。"

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