Integration and Expression of Target Gene in cry2A*-Transgenic Early Japonica Rice with Insect Resistance

TIANChongbing, ZHAOFengmin, SUNHaizheng, SUNShuhong, XUEJingfang, ZHANGXirui, FENGYanjiang, MAWendong, HUYueting

Chin Agric Sci Bull ›› 2026, Vol. 42 ›› Issue (17) : 34-41.

PDF(2195 KB)
Home Journals Chinese Agricultural Science Bulletin
Chinese Agricultural Science Bulletin

Abbreviation (ISO4): Chin Agric Sci Bull      Editor in chief: Yulong YIN

About  /  Aim & scope  /  Editorial board  /  Indexed  /  Contact  / 
PDF(2195 KB)
Chin Agric Sci Bull ›› 2026, Vol. 42 ›› Issue (17) : 34-41. DOI: 10.11924/j.issn.1000-6850.casb2025-0644

Integration and Expression of Target Gene in cry2A*-Transgenic Early Japonica Rice with Insect Resistance

Author information +
History +

Abstract

To investigate the expression characteristics of the target gene in transgenic cry2A* insect resistant early japonica rice, the study employed the T9 generation of transgenic cry2A* early japonica rice line ‘Songjing 9 (cry2A*)’ as experimental material. The transcription levels of the cry2A* gene in various organs at the tillering and heading stages were detected by quantitative real-time PCR (qPCR), and the expression levels of Bt protein in various organs at the tillering, heading and filling stages as well as in brown rice at the full ripening stage were determined by enzyme-linked immunosorbent assay (ELISA).The results showed that the transcription levels of the cry2A* gene varied among the same tissues of different transformation events and among different tissues of the same transformation event. The transcription levels at the heading stage were higher than those at the tillering stage, and the tissue-specific expression pattern was in the order of leaves > young panicles > culm-sheaths. In terms of temporal expression, the Bt protein levels showed the pattern of filling stage > heading stage > tillering stage in leaves and culm-sheaths, and heading stage > filling stage in young panicles; the Bt protein concentration in brown rice was the lowest at the full ripening stage. In terms of spatial expression, the pattern was leaves> culm-sheaths at the tillering stage, leaves>young panicles>culm-sheaths at the heading stage, and leaves > culm-sheaths > young panicles at the filling stage, with the Bt protein levels in all the above tissues consistently higher than those in brown rice at the full ripening stage. In conclusion, the transcription levels of the cry2A* gene in early japonica rice were significantly positively correlated with the Bt protein expression levels, with distinct temporal and spatial variations. Collectively, the expression increased with the progression of growth and development in vegetative organs, while it decreased with the advance of growth stages in reproductive organs.

Key words

cry2A*-transgenic early japonica rice / ‘Songjing 9 (cry2A*)’ / qPCR technology / ELISA technology / Bt protein expression levels / temporal and spatial expression

Cite this article

Download Citations
TIAN Chongbing , ZHAO Fengmin , SUN Haizheng , et al . Integration and Expression of Target Gene in cry2A*-Transgenic Early Japonica Rice with Insect Resistance[J]. Chinese Agricultural Science Bulletin. 2026, 42(17): 34-41 https://doi.org/10.11924/j.issn.1000-6850.casb2025-0644

References

[1]
ZHANG Q. Strategies for developing green super rice[J]. Proceedings of the national academy of sciences of the united states of America, 2007, 104(42):16402-164029.
From a global viewpoint, a number of challenges need to be met for sustainable rice production: (i) increasingly severe occurrence of insects and diseases and indiscriminate pesticide applications; (ii) high pressure for yield increase and overuse of fertilizers; (iii) water shortage and increasingly frequent occurrence of drought; and (iv) extensive cultivation in marginal lands. A combination of approaches based on the recent advances in genomic research has been formulated to address these challenges, with the long-term goal to develop rice cultivars referred to as Green Super Rice. On the premise of continued yield increase and quality improvement, Green Super Rice should possess resistances to multiple insects and diseases, high nutrient efficiency, and drought resistance, promising to greatly reduce the consumption of pesticides, chemical fertilizers, and water. Large efforts have been focused on identifying germplasms and discovering genes for resistance to diseases and insects, N- and P-use efficiency, drought resistance, grain quality, and yield. The approaches adopted include screening of germplasm collections and mutant libraries, gene discovery and identification, microarray analysis of differentially regulated genes under stressed conditions, and functional test of candidate genes by transgenic analysis. Genes for almost all of the traits have now been isolated in a global perspective and are gradually incorporated into genetic backgrounds of elite cultivars by molecular marker-assisted selection or transformation. It is anticipated that such strategies and efforts would eventually lead to the development of Green Super Rice.
[2]
叶高潮, 杜斌, 沈庆雷. 不同药剂防治水稻纵卷叶螟效果研究[J]. 现代农业科技, 2015(2):126.
[3]
王梅, 徐昌文, 宋万勇, 等. 水稻病虫害防治模型及应用[J]. 北京农业, 2015(20):61-62.
[4]
李荣田, 王新宇, 田崇兵, 等. 转cry1C*cry2A*基因早粳稻Bt蛋白的时空表达和抗螟虫性[J]. 作物学报, 2018, 44(12):1829-1836.
早粳稻空育131为受体, 以根瘤农杆菌介导遗传转化法创制了转ubi启动子调控下的cry1C*及cry2A*基因早粳稻空育131 (cry1C*)和空育131(cry2A*)。为了研究转基因水稻Bt蛋白的时空表达特性及抗螟虫性, 将不同转化事件形成的转基因早粳稻品系种植于田间, 利用酶联免疫吸附测定(ELISA)法检测转基因水稻不同生长发育阶段不同器官的、以及成熟期糙米的Bt蛋白量, 采用室内离体茎秆法接虫鉴定转基因水稻的抗螟虫性。结果显示, 转基因早粳稻不同抗虫基因Bt蛋白量不同, cry1C*基因总是低于cry2A*基因的蛋白质表达量; 不同生长发育时期Bt蛋白量不同, 叶片和茎鞘等器官的Bt蛋白量为分蘖期<抽穗期<灌浆期, 幼穗或糙米等器官的Bt蛋白量为抽穗期幼穗>灌浆期幼穗>成熟期糙米; 不同器官Bt蛋白量不同, 高低次序在分蘖期为叶片、茎鞘, 抽穗期为叶片、幼穗和茎鞘, 灌浆及成熟期为叶片、茎鞘、幼穗和糙米; 同一抗虫基因不同转基因品系间抽穗期叶片、茎鞘和幼穗等器官Bt蛋白量及抗螟虫性、糙米Bt蛋白量等性状存在差异, 抽穗期各器官Bt蛋白量与抗螟虫性及成熟期糙米Bt蛋白量之间相关不显著, 不论Bt蛋白量高或低的品系均表现为高抗螟虫。转基因早粳稻营养器官生长发育前期Bt蛋白量较低、后期较高, 繁殖器官生长发育早期Bt蛋白量较高、晚期较低, 营养器官通常比繁殖器官的Bt蛋白量高。在本研究范围内, 不同基因及不同转化事件培育的转基因水稻Bt蛋白表达量高低不同, 但所有的转基因早粳稻品系均表现高抗螟虫。
[5]
唐微. Bt抗虫水稻的培育[D]. 武汉: 华中农业大学, 2006.
[6]
林良斌, 官春云. Bt毒蛋白基因与植物抗虫基因工程[J]. 生物工程进展, 1997(2):50-54.
[7]
NEWELL C A, LOWE J M, MERRYWEATHER A. Transformation of sweet potato with Agrobacterium tumefaciens and regeneration of plants expressing cowpea trypsin inhibitor and snowdrop lectin[J]. Plant science, 1995, 107:215-227.
[8]
GATEHOUSE A M R, DOWN R E, POWELLl K S. Transgenic potato plants with enhanced resistance to the peach-potato aphid Myzus persicae[J]. Entomologia experimentalis et applicata, 2015, 79:295-307
[9]
杨长登, 唐克轩, 吴连斌, 等. 农杆菌介导将雪莲凝集素(GNA)基因转入籼稻单倍体微芽的初步研究[J]. 中国水稻科学, 1998(3):129-133.
以含有双元载体(携带GNA基因,nptⅡ基因)的根癌农杆菌菌系LBA4404转化籼稻单倍体无性系微芽Hu18,经共培养后在含G418的保存培养基中连续筛选G418抗性芽,抗性芽经生根培养基中壮苗获得单倍体转化植株。PCR分析证明GNA基因已进入到Hu18细胞中,抗虫鉴定表明转基因植株具有白背飞虱抗性。并探明了G418对单倍体微芽的致死浓度和时间,共培养时间对G418抗性芽产生的影响。 
[10]
SCHROEDER H E, GOLLASCH S, MOORE A, et al. Bean α-amylase inhibitor confers resistance to the pea weevil (Bruchus pisorum) in transgenic peas (Pisum sativum L.)[J]. Plant physiology, 1995, 107(4):1233-1239.
[11]
王世贵. 转Bt杀虫蛋白基因植物及其抗虫性研究进展[J]. 杭州师范学院学报, 2000(3):90-95.
[12]
ZHU X S, ZHANG T F, ZHU Y X. Cloning and sequencing of two depressant insect selective neurotoxin cDNAs from Buthus martensii Karsch[J]. Chinese science bulletin, 1996, 41:1387-1391.
[13]
PRADO-LOPEZ L, SOBERON M, BRAVO A. Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection[J]. Fems microbiology reviews, 2013, 37(1):3-22.
[14]
RAYMOND B, PAUL R J, DIDIER L. Bacillus thuringiensis: an impo tent pathogen[J]. Trends in microbiology, 2010, 18:189-194.
[15]
ChEN H, TANG W, XU C G, et al. Trans genic indica rice plants harboring a synthetic cry2A* gene of Bacillus thuringiensis exhibit enhanced resistance against lepi dopteran rice pests[J]. Theoretical and applied genetics, 2005, 111(7):1330-1337.
[16]
秦伟, 黄昆仑, 贺晓云, 等. 水稻密码子优化的cry2A*基因在大肠杆菌中的表达及其表达产物的纯化[J]. 食品科学, 2008(7):267-271.
通过PCR从克隆载体pUC18-3Z/Cry2A*上扩增水稻偏爱型密码子优化的抗虫基因cry2A*,经限制性内切酶NdeI和BamHI双酶切定向插入到原核表达载体pET-28a(+),成功构建了在表达蛋白的N端只带有6个组氨酸标签的融合蛋白表达载体pET-28a(+)/Cry2A*,并转入大肠杆菌BL21(DE3)中。通过对其表达条件进行优化,发现在IPTG浓度为0.05mmol/L、诱导时间为3h、诱导温度为20℃的表达条件下目的蛋白大部分以可溶形式进行表达。采用Ni-NTA亲和柱纯化得到高纯度目的蛋白,薄层扫描分析蛋白纯度达到95%。
[17]
吴振映, 柳絮, 王文英, 等. 利用农杆菌介导法获得转cry2A抗虫基因水稻的研究[J]. 山东农业科学, 2011(5):1-3,7.
[18]
矫江, 李禹尧, 中本和夫. 黑龙江省水稻生产发展区划研究[J]. 黑龙江农业科学, 2012(11):1-4.
[19]
李艳君. 水稻二化螟在五常市的发生规律及防治技术探讨[J]. 中国农村小康科技, 2010(7):67-68.
[20]
CRICKMORE N, ZEIGLER N R, FEITELSON J, et al. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins[J]. Microbiology and molecular biology reviews, 1998, 62(3):807-813.
The crystal proteins of Bacillus thuringiensis have been extensively studied because of their pesticidal properties and their high natural levels of production. The increasingly rapid characterization of new crystal protein genes, triggered by an effort to discover proteins with new pesticidal properties, has resulted in a variety of sequences and activities that no longer fit the original nomenclature system proposed in 1989. Bacillus thuringiensis pesticidal crystal protein (Cry and Cyt) nomenclature was initially based on insecticidal activity for the primary ranking criterion. Many exceptions to this systematic arrangement have become apparent, however, making the nomenclature system inconsistent. Additionally, the original nomenclature, with four activity-based primary ranks for 13 genes, did not anticipate the current 73 holotype sequences that form many more than the original four subgroups. A new nomenclature, based on hierarchical clustering using amino acid sequence identity, is proposed. Roman numerals have been exchanged for Arabic numerals in the primary rank (e.g., Cry1Aa) to better accommodate the large number of expected new sequences. In this proposal, 133 crystal proteins comprising 24 primary ranks are systematically arranged.
[21]
SHU C L, ZHANG F J, CHEN G H, et al. A natural hybrid of a Bacillus thuringiensis Cry2A toxin implicates domain I in specificity determination[J]. Journal of invertebrate pathology, 2017, 150:35-40.
[22]
刘洋, 柳青, 李楠, 等. 转cry1Ab/Gc基因玉米的不同转化事件Bt蛋白表达和抗虫性分析[J]. 哈尔滨师范大学(自然科学学报), 2016, 32(3):87-92.
[23]
夏兰芹, 徐琼芳, 郭三堆. 抗虫棉生长发育过程中Bt杀虫基因及其表达的变化[J]. 作物学报, 2005(2):197-202.
[24]
汪秀峰, 叶芬, 李莉, 等. 转基因水稻恢复系及其F1代Bt蛋白的时空表达分析[J]. 分子植物育种, 2014, 12(6):1077-1081.
[25]
于志晶, 蔡勤安, 林秀峰, 等. 转基因抗虫水稻中Bt蛋白表达量的研究[J]. 安徽农业科学, 2012, 40(6):3251-3252.
[26]
朱路青, 曹越平. 转Bt基因大豆植株中Bt毒蛋白的表达[J]. 上海交通大学学报(农业科学版), 2005(3):234-238.
[27]
CHOI S B, WANG C, MUENCH D G, et al. Messenger RNA targeting of rice seed storage proteins to specific ER subdomains[J]. Nature. 2000, 407(6805):765-767.
[28]
REN Y L, WANG Y H, LIU F, et al. Glutelin precursor accumulation3 en- codes a regulator of post-Golgi vesicular traffic essential for vacuolar protein sorting in rice endosperm[J]. Plant cell, 2014, 26:410-425.
In seed plants, a major pathway for sorting of storage proteins to the protein storage vacuole (PSV) depends on the Golgi-derived dense vesicles (DVs). However, the molecular mechanisms regulating the directional trafficking of DVs to PSVs remain largely elusive. Here, we report the functional characterization of the rice (Oryza sativa) glutelin precursor accumulation3 (gpa3) mutant, which exhibits a floury endosperm phenotype and accumulates excess proglutelins in dry seeds. Cytological and immunocytochemistry studies revealed that in the gpa3 mutant, numerous proglutelin-containing DVs are misrouted to the plasma membrane and, via membrane fusion, release their contents into the apoplast to form a new structure named the paramural body. Positional cloning of GPA3 revealed that it encodes a plant-specific kelch-repeat protein that is localized to the trans-Golgi networks, DVs, and PSVs in the developing endosperm. In vitro and in vivo experiments verified that GPA3 directly interacts with the rice Rab5a-guanine exchange factor VPS9a and forms a regulatory complex with Rab5a via VPS9a. Furthermore, our genetic data support the notion that GPA3 acts synergistically with Rab5a and VPS9a to regulate DV-mediated post-Golgi traffic in rice. Our findings provide insights into the molecular mechanisms regulating the plant-specific PSV pathway and expand our knowledge of vesicular trafficking in eukaryotes.
PDF(2195 KB)

Accesses

Citation

Detail

Sections
Recommended

/