The ability of various pests and diseases to adapt to a single plant resistance gene over time leads to loss of resistance in transgenic rice. Therefore, introduction of different pest and disease resistance genes is critical for successful cultivation of transgenic rice strains with broad-spectrum resistance to multiple pathogens. Here, we produced resistance rice lines with multiple, stacked resistance genes by stacking breeding and comprehensively evaluated their resistance to Chilo suppressalis (striped rice stemborer), Magnaporthe oryzae (rice blast), and Nilaparvata lugens (brown planthopper) in a pesticide-free environment. CRY1C and CRY2A are exogenous genes from Bacillus thuringiensis. Pib, Pikm, and Bph29 are natural genes in rice. CH121TJH was introduced into CRY 1C, Pib, Pikm, and Bph29. CH891TJH and R205XTJH were introduced into CRY 2A, Pib, Pikm, and Bph29. Compared with those observed in their recurrent parents, CH121TJH significantly increased the mortality of borers. The other two lines CH891TJH and R205XTJH are the same result. Three lines introduction of Pib and Pikm significantly reduced the area of rice blast lesions, and introduction of Bph29 significantly reduced seedling mortality from N. lugens. Introduction of the exogenous genes had relatively few effects on agronomic and yield traits of the original parents. These findings suggest that stacking of rice resistance genes through molecular marker-assisted backcross breeding can confer broad spectrum and multiple resistance in differently genetic backgrounds.

The extent of molecular diversity and differentially expressed proteins (DEPs) in transgenic lines provide valuable information to understand the phenotypic performance of transgenic crops compared with their parents. Here, we compared the differences in the phenotypic variation of twelve agronomic and end-use quality traits, the extent of microsatellite diversity, and DEPs of a recurrent parent line with three transgenic rice restorer lines carrying either CRY1C gene on chromosome 11 or CRY2A gene on chromosome 12 or both genes. The three transgenic lines had significantly smaller stem borer infestation than the recurrent parent without showing significant differences among most agronomic traits, yield components, and end-use quality traits. Using 512 microsatellite markers, the three transgenic lines inherited 2.9-4.3% of the Minghui 63 donor genome and 96.3-97.1% of the CH891 recurrent parent genome. As compared with the recurrent parent, the number of upregulated and down-regulated proteins in the three transgenic lines varied from 169 to 239 and from 131 to 199, respectively. Most DEPs were associated with the secondary metabolites biosynthesis transport and catabolism, carbohydrate transport and metabolism, post-translational modification, and signal transduction mechanisms. Although several differentially expressed proteins were observed between transgenic rice and its recurrent parent, the differences may not have been associated with grain yield and most other phenotypic traits in transgenic rice.

A lack of stability in the expression of Bacillus thuringiensis genes (CRY) and the dialaninophosphate resistance gene (BAR) in transgenic rice plants can lead to the loss of important characters. The genetic stability of transgenic expression in high-generation lines is thus critically important for ensuring the success of molecular breeding efforts. Here, we studied the genetic stability of resistance to insect pests and herbicides in transgenic rice lines at the molecular and phenotypic levels in a pesticide-free environment. Southern blot analysis, real-time polymerase chain reaction, and enzyme-linked immunosorbent assays revealed high stability in the copy numbers and expression levels of CRY1C, CRY2A, and BAR in transgenic lines across different generations, and gene expression levels were highly correlated with protein expression levels. The insecticide resistance of the transgenic rice lines was high. The larval mortality of Chilo suppressalis was 50.25% to 68.36% higher in transgenic lines than in non-transgenic control lines. Percent dead hearts and percent white spikelets were 16.66% to 22.15% and 27.07% to 33.47% lower in transgenic lines than in non-transgenic control lines, respectively. The herbicide resistance of the transgenic rice lines was also high. The bud length and root length ranged were 2.53 cm to 4.20 cm and 0.28 cm to 0.73 cm higher in transgenic lines than in non-transgenic control lines in the budding stage, respectively. Following application of the herbicide Basta, the chlorophyll content of the transgenic lines began to recover 2 d later in the seedling and tillering stages and 3 d later in the booting and heading stages, by contrast, the chlorophyll content of the non-transgenic lines did not recover and continued to decrease. These findings revealed high genetic stability of the resistance to insect pests and herbicides across several generations of transgenic rice regardless of the genetic background.

Abstract Omics techniques provide effective detection tools for assessing the potential impact of plant composition at the DNA, RNA, and protein levels. Among these, protein is the executor of gene function and the embodiment of biological traits, so that organisms show various genetic characteristics. Proteomics can be used to assess whether genetic engineering will lead to changes in plant traits beyond those introduced by conventional plant breeding. Here, we compare the extent of the proteome occurring in the leaves of three transgenic rice restore lines expressing CRY1C and CRY2A genes developed by genetic engineering and their corresponding recurrent parents developed by conventional breeding. CRY1C and CRY2A genes were inserted into chromosomes 11 and 12, respectively, which significantly improved the resistance of restore lines to Chilo suppressalis. Although differentially expressed proteins could be distinguished between transgenic rice and its recurrent parents, these differences were not sufficient to cause unintended effects on grain yield and quality traits of transgenic rice. In contrast, differences in phenotypic traits are more because of differences in genetic background. Functional cluster analysis showed that the differentially expressed proteins caused by the insertion of exogenous genes did not involve harmful metabolic pathways. The study successfully used 4D label-free quantitative proteomics technology to assess the unexpected changes in new rice varieties, and the results showed that transgenic rice did not cause unintended effects.

The insect resistance of Bacillus thuringiensis (Bt) transgenic rice (Oryza sativa L.) is mainly determined by the transcription of the CRY1C gene and the translation process of the Cry1C protein. With this in mind, we analyzed CRY1C expression and Cry1C protein content in a transgenic Bt insect-resistant restorer line and its F1 hybrids, evaluated the resistance to Chilo suppressalis of various lines of rice, and determined the 50% lethal concentration (LC50) of Cry1C for C. suppressalis. In four transgenic rice lines, the relative expression of CRY1C was highest at the heading stage in most tissues. Among different tissues from the same developmental stage, CRY1C expression was highest in leaves, followed by stems and panicles. The relative expression of CRY1C was higher in the parent restorer line than in the F1 hybrids. The LC50 of the Cry1C protein for C. suppressalis was 4.016 mu g g(-1), and the Cry1C protein expression level of transgenic insect-resistant rice exceeded this threshold at the heading stages and in stems. We next analyzed the lethality of each strain toward C. suppressalis. After 48 h, the mortality rate of second-instar C. suppressalis larvae feeding on transgenic stem tissue was higher at the heading stage than at the tiller stage and varied from 66.7 to 91.7%. By documenting the temporal and spatial expression of CRY1C and evaluating C. suppressalis resistance to Bt transgenic rice, the risk of pests and diseases during the rice production process can be greatly reduced.

使用5个转Bt基因水稻恢复系作为父本,以12个不育系作为母本,根据5×12(NCII)不完全双列杂交设计配制60个组合,分析了这些组合的9个稻米品质性状的配合力及遗传参数.结果表明:精米率主要受基因加性效应的影响,整精米率、垩白粒率、碱消值、胶稠度主要受基因非加性效应互作的影响;除精米率主要受母本影响外,其余8个性状均受双亲交互作用的影响;垩白粒率和垩白度在后代遗传中主要受基因遗传作用的影响,可以在早代直接选择.配合力方差分析结果表明:精米率、垩白度及碱消值受环境因素的影响较大;昌恢891T为一般配合力较好的父本,华1165S为一般配合力较好的母本;原香39A/昌恢891T、泰乡1209A/昌恢T025T、昌盛843A/昌恢T025T为较优的组合,其中原香39A/昌恢891T的特殊配合力的综合评价最好.

Yazhan is a restorer rice line bred by Jiangxi Tianya Seed Industry Co., LTD, and its hybrid combination has a high yield, excellent rice quality, and strong resistance. In this study, Yazhan, Huazhan, and Zhanhui 15 were used as the experimental materials. Based on agronomic traits, SSR primer screening and second-generation resequencing were conducted to study the genetic basis of Yazhan. The results showed that the plant height of Yazhan was significantly lower than Zhanhui 15, and the yield was not different from the parent lines. The genetic material on chromosome 1, 3, 4, 5, and 10 in the genome of Yazhan were mainly from Zhanhui 15, genetic materials from chromosomes 2, 6, 8, 9, 11, and 12 mainly come from Huazhan, and the chromosomes 1, 5, and 6 had the most variation. In terms of genomic similarity, Yazhan was more similar to the male parent (Huazhan). Yazhan and Huazhan carried the Wxb genotype, while Zhanhui 15 carried the Wxa genotype. The amylose contents of Yazhan and Huazhan were significantly lower than Zhanhui 15. Yazhan and Huazhan carried the Pita blast resistance gene, and Zhanhui 15 carried the Pita susceptibility gene. The results of this study provide a theoretical reference for elucidating the genetic composition of Yazhan and determining the main genetic sources of its important agronomic traits.

4个稳定转Bt基因恢复系和6个不育系组成4×6不完全双列杂交,对8个主要产量农艺性状进行了配合力分析.结果表明,恢复系和不育系各产量农艺性状的配合力方差均达到显著差异.一般配合力(GCA)表明昌恢T025 T、R205选T、C815S、乡泰A的GCA配合力高.特殊配合力(SCA)效应分析表明C815S/昌恢121T、乡泰A/R205选T、野香A/昌恢T025T、乡泰A/昌恢T025T的SCA表现好,这4个F1后代是单株产量优良的杂交组合.基因型方差贡献率分析表明结实率、千粒质量主要受基因加性效应影响,有效穗数、穗长、实粒数、总粒数、单株产量主要受特殊配合力(SCA)的影响.实粒数、总粒数、单株产量主要受母本影响,其他性状则由父母本共同决定.有效分蘖、总粒数的狭义遗传率较低,受遗传因素影响较小,要在晚代或间接选择才有效.

研究杂交组合的遗传特性,挖掘优异的种质资源对于杂种优势利用具有重要意义.本试验以8个恢复系为父本、21个不育系为母本,按照不完全双列杂交8×21(NCII)设计配制168个组合,对8个农艺性状的配合力及遗传参数进行分析.结果 表明:有效穗数、穗长、千粒重主要受基因加性效应影响,株高、实粒数、结实率主要受基因非加性效应互作影响;杂交组合的株高、有效穗数、穗长、千粒重、单株产量的表现主要依赖于母本,总粒数受父本影响更大,实粒数、结实率取决于双亲的表现;有效穗数、单株产量在后代遗传中的稳定性较差,易受环境和基因非加性效应影响;843A、宜香1A、沪旱7A、昌恢871、昌恢T025、雅占为配合力好的亲本,宜香1A、昌恢T025具有最好的一般配合力(GCA)效应值;宜香1A/昌香恢1号、千乡059A/昌恢121、广8A/昌恢881为较优组合,宜香1A/昌香恢1号特殊配合力(SCA)最优.对恢复系主要农艺性状进行配合力分析,为杂交水稻恢复系的选育提供了一定的科学依据.

转基因水稻中外源基因的转入可能会形成新的代谢产物,造成农艺性状的改变等,这些均不利于农业生产的非预期效应.为探究转基因水稻是否存在非预期效应,本研究以江西农业大学选育的4个转Bt基因恢复系为试验材料,通过反向PCR扩增侧翼序列并比对,用216对SSR引物对供试材料进行全基因组背景分析,再以Real-time PCR检测外源基因插入位点上下游100 kb内所有基因表达差异,并对光合色素含量及9个主要农艺性状进行显著性分析.结果表明,cry1C侧翼序列片段大小为1 276bp,定位在水稻第11号染色体上,cry2A侧翼序列片段大小为948 bp,定位在水稻第12号染色体上;昌恢121T、昌恢891T、昌恢T025T、R205选T的遗传背景回复率分别为95.60％、88.43％、93.52％、94.44％;基因表达分析显示试验组和对照组在cry1C、cry2A插入位置上下游100 kb内所有侧翼基因的表达水平均无显著性差异;进一步调查发现,试验组和对照组的光合色素含量及9个农艺性状无显著性差异.综上,cry1C和cry2A插入水稻基因组没有改变侧翼100 kb内基因表达水平,且4个转基因水稻并未产生非预期效应.本研究结果为探究转基因水稻的非预期效应评价提供了一定的理论参考.

以5个新育成的转Bt基因抗虫恢复系为父本,7个不育系为母本,采用NCII不完全双列杂交配制35个组合,对9个主要农艺性状进行了配合力分析.配合力方差分析表明亲本的一般配合力方差均高于组合的特殊配合力方差,说明在决定组合的农业性状上亲本的加性效应占了主导作用.亲本的一般配合力(GCA)效应分析表明洪A、荃9311A、昌恢121T、昌恢T025T的GCA效应值高,这4个亲本的配合力高.杂交组合的特殊配合力(SCA)效应分析表明,荃9311A/昌恢T025T的9个农艺性状综合SCA效应值最好;67A/R205T的单株产量SCA效应值最高;荃9311A/昌恢606T具有最小的株高SCA效应值;穗长SCA效应值最大的是843A/昌恢891T;843A/昌恢121T的空批粒数SCA效应值最低,结实率SCA最高.综上所述,荃9311A/昌恢T025T、67A/R205T、洪A/昌恢121T、843A/昌恢121T表现好.