已入深夜,您辛苦了!由于当前在线用户较少,发布求助请尽量完整地填写文献信息,科研通机器人24小时在线,伴您度过漫漫科研夜!祝你早点完成任务,早点休息,好梦!

Enhancing peanut nutritional quality by editing AhKCS genes lacking natural variation

生物 花生 种质资源 基因 油酸 食品科学 脂肪酸 花生油 生物技术 生物化学 植物 生态学 原材料
作者
Dongxin Huai,Xiaomeng Xue,Jie Wu,Manish K. Pandey,Nian Liu,Li Huang,Liying Yan,Yuning Chen,Xin Wang,Li Wang,Yanping Kang,Zhihui Wang,Huifang Jiang,Rajeev K. Varshney,Boshou Liao,Yong Lei
出处
期刊:Plant Biotechnology Journal [Wiley]
标识
DOI:10.1111/pbi.14423
摘要

Peanut (Arachis hypogaea L.) is a globally staple oilseed crop, extensively cultivated in tropical and subtropical regions. Due to its substantial oil (approximately 46%–58%) and protein (around 22%–32%) content, the peanut plays a pivotal role in addressing malnutrition and ensuring food security in many regions. The fatty acid profiles of vegetable oil and foods have recently garnered increased attention due to the potential impact on human health. Very long chain fatty acids (VLCFAs) are defined as fatty acids with a carbon chain length exceeding 18 atoms (Guyomarc'h et al., 2021). Peanut kernels contain various VLCFAs, such as arachidic acid (C20:0), eicosenoic acid (C20:1), behenic acid (C22:0) and lignoceric acid (C24:0), but most of them are saturated fatty acids (SFAs). It is well understood that high levels of very long chain saturated fatty acid (VLCSFA) are associated with prevalence of atherosclerosis and cardiovascular disease (Bloise et al., 2022). Therefore, reducing the VLCFA content in peanuts has gained more importance realizing its positive impact for improving the nutritional quality and health value. The biosynthesis of VLCFAs in plants is known to be regulated by a key enzyme, β-ketoacyl-CoA synthase (KCS) (Wang et al., 2017). In our previous study, a total of 30 AhKCS genes were identified in peanut genomes. After gene expression profiling and functional analysis, a pair of homologous gene AhKCS1 and AhKCS28 were identified as putative regulators of VLCFA contents in peanut kernels. The VLCFA content in available peanut germplasm accessions ranges from 4.3% to 9.8%, but no sequence variation was observed within or surrounding the AhKCS1 and AhKCS28 genes, suggesting the only possibility of further reduction of VLCFA content through gene editing (Huai et al., 2020). Therefore, in this study, AhKCS1 and AhKCS28 were genetically disrupted using the CRISPR/Cas9 system to generate novel peanut mutants exhibiting significantly reduced levels of VLCFA content in kernels. A CRISPR/Cas9 construct was designed to incorporate two single-guide RNAs (sgRNAs) that specifically target the homologous exon regions of AhKCS1 and AhKCS28 genes (Figure 1a,b). Firstly, this construct was introduced into normal oleate peanut cultivar Zhonghua 12 (ZH12) through Agrobacterium tumefaciens-mediated transformation (Huai et al., 2023). A total of 66 independent positive T0 transgenic ZH12 plants were successfully obtained. Among them, 61 exhibited mutations in both target genes, while two showed mutations in only one gene (Table S1). Three homozygous T1 lines (A-2, A-3 and A-9) with mutations at both target sites for sgRNA1 and sgRNA2 in AhKCS1 and AhKCS28 genes, which caused translational frameshifts and premature stop codons, were selected for further study (Figures 1b and S1). None of the AhKCS1/AhKCS28 double mutants exhibited any growth anomalies, and no apparent alteration in morphological and yield-related traits under both greenhouse and field conditions. Furthermore, resequencing of the three double mutants revealed no evidence of off-target mutations (Table S2). The fatty acid composition of the harvested seeds from ZH12 and each double mutant was determined by gas chromatography (Figure 1c). The VLCFAs contents in the double mutants have been significantly decreased by 70.6%–100.0%. The VLCFA profiles of ZH12 showed four distinct peaks corresponding to C20:0, C20:1, C22:0 and C24:0. However, the peak of C20:1 and C24:0 was absent in all the three double mutants (Figure 1c). Although the peak of C20:0 was observed in both ZH12 and the double mutants, its content significantly decreased from 1.7% to 0.4%–0.5% in the double mutants. Similarly, while the content of C22:0 amounted to 2.8% in ZH12, it dramatically reduced to 0.3% in A-2 and was absent altogether in A-3 and A-9. Consequently, there was a substantial reduction from total VLCFA content of 6.9% observed within ZH12 down to merely 0.9%, 0.5% and 0.4% in A-2, A-3 and A-9, respectively, which were considerably lower than the value (4.3%) in naturally evolved germplasm materials (Figure 1d). The CRISPR/Cas9 construct was also introduced into a high oleate peanut breeding line JC30. In total, 63 independent positive T0 transgenic JC30 plants were generated, out of which 60 exhibited mutations in both target genes (Table S1). Similarly, three homozygous T1 lines (B-37, B-38 and B-59) harbouring truncated proteins of AhKCS1 and AhKCS28 were chosen to analyse the seed fatty acid composition (Figures 1b and S1). The double mutants of JC 30 exhibited only three peaks representing to C20:0, C20:1 and C22:0, while the peak of C24:0 was not detected (Figure 1c). The contents of C20:0 and C20:1 in double mutants of JC30 were reduced from 1.0% to 0.4%, while the C22:0 content was decreased from 1.4% to 0.2%. The VLCFA content in the double mutants of JC30 was reduced from 4.1% to 1.0%, which was slightly higher than that of double mutants of ZH12 (0.4%–0.9%). This relatively higher content can be attributed to the higher C20:1 content in the double mutants of JC30, which was absent in the double mutants of ZH12 (Figure 1d). The increase of C20:1 in double mutants of JC30 can be explained by an augment availability of substrate C18:1 in kernels. Interestingly, there was no significant difference in total VLCSFA content between the double mutants derived from JC30 and ZH12 (0.6%–0.7% vs 0.4%–0.9%). Additionally, the levels of C16:0, C18:0 and C18:2 were found to be elevated, while the content of C18:1 was observed to be slightly reduced in both double mutants derived from JC30 and ZH12 (Figure 1c,d). In summary, we demonstrated that AhKCS1 and AhKCS28 genes with no natural variation are the key genes for controlling the seed VLCFA content in peanut, and developed novel germplasm lines with low seed VLCFA content using genome-editing system. Furthermore, we also provided an efficient CRISPR/Cas9 genome editing platform for peanut, with great potential for expediting breeding programmes aimed at improving traits such as yield, quality and stress resistance. This work was supported by the Key Research and Development Program of China (2023YFD1202800), the Knowledge Innovation Program of Wuhan-Basic Research (2022020801010291), the Project of the Development for High-quality Seed Industry of Hubei province (HBZY2023B003) and Innovation Program of the Chinese Academy of Agricultural Sciences (2023-2060299-089-031). DH, RKV, BL and YL conceived and designed the experiments; HJ and LH supplied the peanut cultivars; XX, JW, NL, LY, YC, XW, QW, YK and ZW performed the experiments; DH, XX and MKP analysed the data; DH wrote the manuscript; DH, MKP, RKV, BL and YL contributed in data interpretation and revision of the manuscript. All authors have read and approved the final version of the manuscript. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Figure S1. The Sanger sequencing chromatograms of each target site in the homozygous T1 lines. Table S1. Summary of mutations at each target site in the T0 generation. Table S2. Detection of off-target mutation in A-2, A-3 and A-9 using genome resequencing. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
最长约 10秒,即可获得该文献文件

科研通智能强力驱动
Strongly Powered by AbleSci AI
更新
PDF的下载单位、IP信息已删除 (2025-6-4)

科研通是完全免费的文献互助平台,具备全网最快的应助速度,最高的求助完成率。 对每一个文献求助,科研通都将尽心尽力,给求助人一个满意的交代。
实时播报
Karol完成签到,获得积分10
1秒前
小小完成签到 ,获得积分10
3秒前
简单山水完成签到,获得积分10
7秒前
小二郎应助刺猬采纳,获得10
7秒前
14秒前
14秒前
14秒前
刺猬完成签到,获得积分10
15秒前
烟花应助简单山水采纳,获得10
17秒前
烟花应助认真的三问采纳,获得10
20秒前
22秒前
andrele发布了新的文献求助10
22秒前
权小夏完成签到 ,获得积分10
23秒前
吴嘉俊完成签到 ,获得积分10
26秒前
29秒前
轻松的惜芹应助aliu采纳,获得10
31秒前
33秒前
CC发布了新的文献求助10
34秒前
boymin2015完成签到 ,获得积分10
34秒前
生物科研小白完成签到 ,获得积分10
41秒前
LMX完成签到 ,获得积分10
41秒前
chenlc971125完成签到 ,获得积分10
41秒前
44秒前
44秒前
IfItheonlyone完成签到 ,获得积分10
53秒前
SHD完成签到 ,获得积分10
54秒前
yu完成签到 ,获得积分10
55秒前
55秒前
Mr兔仙森完成签到,获得积分10
59秒前
悠悠我心完成签到,获得积分10
1分钟前
英姑应助霜鸣采纳,获得10
1分钟前
1分钟前
凶狠的寄风完成签到 ,获得积分10
1分钟前
1分钟前
水若琳发布了新的文献求助10
1分钟前
小黑完成签到,获得积分10
1分钟前
1分钟前
壮观的谷冬完成签到 ,获得积分0
1分钟前
霸气的思柔完成签到,获得积分10
1分钟前
aliu完成签到,获得积分10
1分钟前
高分求助中
A new approach to the extrapolation of accelerated life test data 1000
ACSM’s Guidelines for Exercise Testing and Prescription, 12th edition 500
Indomethacinのヒトにおける経皮吸収 400
Phylogenetic study of the order Polydesmida (Myriapoda: Diplopoda) 370
基于可调谐半导体激光吸收光谱技术泄漏气体检测系统的研究 350
Robot-supported joining of reinforcement textiles with one-sided sewing heads 320
Aktuelle Entwicklungen in der linguistischen Forschung 300
热门求助领域 (近24小时)
化学 材料科学 医学 生物 工程类 有机化学 生物化学 物理 内科学 纳米技术 计算机科学 化学工程 复合材料 遗传学 基因 物理化学 催化作用 冶金 细胞生物学 免疫学
热门帖子
关注 科研通微信公众号,转发送积分 3989972
求助须知:如何正确求助?哪些是违规求助? 3532034
关于积分的说明 11256042
捐赠科研通 3270884
什么是DOI,文献DOI怎么找? 1805093
邀请新用户注册赠送积分活动 882256
科研通“疑难数据库(出版商)”最低求助积分说明 809216