亚洲免费av电影一区二区三区,日韩爱爱视频,51精品视频一区二区三区,91视频爱爱,日韩欧美在线播放视频,中文字幕少妇AV,亚洲电影中文字幕,久久久久亚洲av成人网址,久久综合视频网站,国产在线不卡免费播放

        ?

        Improved Eating and Cooking Quality of indica Rice Cultivar YK17 via Adenine Base Editing of Wxa Allele of Granule-Bound Starch Synthase I (GBSS I)

        2021-08-31 02:13:50MahmudaBinteMonsurCaoNiWeiXiangjinXieLihongJiaoGuiaiTangShaoqingNeseSreenivasuluShaoGaonengHuPeisong
        Rice Science 2021年5期

        Mahmuda Binte Monsur, Cao Ni, Wei Xiangjin, Xie Lihong, Jiao Guiai, Tang Shaoqing, Nese Sreenivasulu, Shao Gaoneng, Hu Peisong

        Letter

        Improved Eating and Cooking Quality ofRice Cultivar YK17 via Adenine Base Editing ofWxAllele of Granule-Bound Starch Synthase I (GBSS I)

        Mahmuda Binte Monsur1, #, Cao Ni1, #, Wei Xiangjin1, Xie Lihong1, Jiao Guiai1, Tang Shaoqing1, Nese Sreenivasulu2, Shao Gaoneng1, Hu Peisong1

        (State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China; Applied Functional Genomics Cluster, Grain Quality and Nutrition Centre, Strategic Innovation Platform, International Rice Research Institute, Los Banos 4030, the Philippines; These authors contributed equally to this work)

        Amylose content (AC) is the key determinant of eating and cooking quality (ECQ) of rice. The majorWxallele of granule-bound starch synthase I (GBSS I) inrice produces higher AC, making rice hard and dry after cooking. Recent work has improved ECQ ofrice via clustered regularly interspaced short palindromic repeats/CRISPR- associated protein 9 (CRISPR/Cas9) or cytosine base editing (CBE) techniques. However, base editing has not yet been applied to modify theWxallele ofrice. We utilized a novel precise adenine base editing (ABE) tool to generate three mutants ofcultivar Zhongjiazao 17 (YK17) with reduced AC while other ECQ parameters, such as gel consistency (GC) and alkali spreading value, were maintained. Our study demonstrated improvement of ECQ ofrice and will help rice breeders satisfy consumers.

        AC, which determines ECQ in rice, is mainly controlled by(), a gene encoding GBSS I (Teng et al, 2012; Zhang et al, 2021). Variation at thelocus is largely responsible for the diversity of AC levels (Tian et al, 2009; Biselli et al, 2014; Zhang et al, 2019). Alleles includingWx,Wx,Wx,Wx,Wx,Wx,Wx,Wxandhave been reported (Cai et al, 1998; Sato et al, 2002; Larkin and Park, 2003; Wanchana et al, 2003; Mikami et al, 2008; Liu et al, 2009; Yang et al, 2013; Li et al, 2020; Zhang et al, 2021). Higher AC and lower GC values correlate with poor taste due to hard texture, whereas moderate AC rice (15%–20%) with higher GC values (60–80 mm) leading to soft texture is preferred by most consumers worldwide (Zeng et al, 2020). Invarieties, alleleWxgives rise to 25%–30% AC (Wang et al, 1995), while invarieties alleleWxproduces 15%–18% AC (Zhang et al, 2018). The ‘Old’ natural and ‘New’ editedalleles in rice crops will be helpful in developing novel rice varieties and for further quality improvement (Huang et al, 2020a).

        Traditional successive backcrossing methods have been used to improve ECQ by introgressingWx, with moderate AC, intorice. However, traditional breeding strategies are always time-consuming and difficult to break close linkages with unfavourable traits. Recently, CRISPR/Cas9-based genomic editing techniques have been widely used for editing thegene of rice. The glutinous rice was generated by CRISPR/ Cas9-targeted mutagenesis of thegene in elite rice varieties (Zhang et al, 2018; Fei et al, 2019). Moreover, novelalleles with fine-tuned amylose levels can be created and rice grain quality can be improved by promoter editing using CRISPR/Cas9 (Huang et al, 2020b). Zeng et al (2020) reported thequantitative regulation ofexpression by CRISPR/Cas9-based promoter and 5-UTR-intron editing, improving grain quality in rice. Recently, base editing is an advanced CRISPR-based tool that ensures base conversion in a target gene (Komor et al, 2016; Gaudelli et al, 2017; Molla and Yang, 2019; Monsur et al, 2020). CBE, which converts cytosine (C) to thymine (T), has been applied successfully to fine-tune AC inrice (Xu et al, 2020). Modifying theWxallele using base editing strategies has the potential to generaterice with desirable AC to weak the textural preferences.

        In this study, we focused on improving ECQ ofrice cultivars by decreasing AC via a precise ABE tool that can convert adenine (A) to guanine (G) (Li et al, 2018). We selectedrice cultivar YK17 as the super rice cultivar of China with high seed-setting rate and yield, early maturation, moderate plant morphology and good disease resistance (Barman et al, 2019). However, it produces high AC (27.3%) owning to theWxallele (Fig. S1). In our work, 10 target sites were selected randomly in the conserveddomain with glycosyl transferase activity supported by CRISPRdirect (http://crispr.dbcls.jp/) and CRISPR-GE (http://skl.scau.edu.cn/). The sgRNAs were modified into enhanced sgRNA (esgRNA) to make an optimal form for plant ABE-7 (PABE-7). Using the adenosine deaminase, nuclear localization sequences PABE-7 and esgRNA, Li et al (2018) created the efficient vector pH- PABE-7-esgRNA. The constructed plasmids were individually transformed into YK17 by the- mediated method. Independent transgenic plants were generated for each transformant and all the target sequences were sequenced. Target sites on exons 2, 6 and 8 ofWxhad mutations (Fig. 1-A and Table S1). Mutation efficiency varied from 37.50% to 69.23% in T0generation (Table S2). Sequencing results showed that homozygous mutants in three target sites, hereafter named as transgenicto(,and), were obtained in T0generation (Fig. S2), which were further used to generate the T1plants. T-DNA segregation confirmation of T0and T1plants was conducted throughselection on media (Fig. S3). Only T-DNA free plants were used for further studies (Fig. 1-B to -E). Inand, base conversion of A to G resulted in asparagine (AC) conversion to aspartate (AC), whereas in, glutamine (CG) converted to arginine (CG) (Fig. 1-A). Also, the positions of varied nucleotide (1246, 1634 and 496) and amino acid (247, 306 and 128) in consistent with three mutants are showed in Fig. 1-A. Multiple sequence alignment reflected that the mutated amino acid inwas less conserved in comparison to those inand(Fig. S4). Furthermore, five putative off-target sites were screened for each target site and no off-target effects were detected in all the T-DNA-free plants by the DNA sequencing method (Table S3).

        Brown rice from wild type YK17 had a chalkiness appearance, as didand, whereashad opaque phenotypic appearance (Fig. 1-F to -I). For confirmation, we measured the AC and found(25.6%),(16.7%) and(3.4%) had lower AC than that of wild type YK17 (27.3%) (Fig. 1-J), which was consistent with the conclusion that the varied amino acids is less conserved incompared toand. Generally, based on AC values, rice grain is classified into five groups: waxy (0%–5%), very low (5%– 12%), low (12%–20%), intermediate (20%–25%) and high (25%–33%) groups (Juliano, 1998; Zhang et al, 2018). Notably,had low AC, whereashad AC as low as glutinous rice (Fig. 1-J) and fell into the waxy category. GC values decreased slightly in(76 mm) and(77 mm) and increased in(90 mm) compared to YK17 (81 mm) (Fig. 1-K). Thus, GC values forandare in the desirable range (60–80 mm). Alkali spreading value, which stands for gelatinization temperature (GT), did not differ significantly between the wild type and mutants (Fig. 1-L). Those results indicated that the amino acid substitutions in Waxy led to ECQ alterations. In addition, we used rapid visco analysis to evaluate grain starch quality. A decreasing trend of viscosity was observed with decreasing AC (Fig. 1-M). Further, we checked total GBSS I via western blot. Compared to YK17, the mutations inandled to increased GBSS I accumulation in 10-day filling grains (Fig. 1-N), especially in, probably caused by the reduction of Waxy protein degradation. Agronomic traits like grain length, grain width and grain thickness were also measured, and most phenotypes increased in the mutants compared to wild type. However, 1000-grain weight increased only in(Table S4).

        Fig. 1. Desirable amylose content ofrice cultivar YK17 via adenine base editing ofgene.

        A, Structure of Wxand the mutations in edited T1lines. Protospacer-adjacent motifs (PAMs) include three bases (NGG) with a redunderline. The red letters indicate altered bases. Q, Glutamine; R,Arginine; N, Asparagine; D, Aspartate. B–E, Gross morphologies of the wild type YK17 and its mutants,and. Scale bars are 10 cm. F–I, Appearance and transverse sections of brown rice of YK17 and mutants. Scale bars are 1 mm. J, Amylose content. K, Gel consistency. L, Alkali spreading values of the wild type and mutants. Data are Mean ± SD (= 3) in J–L. Samples with different lowercase letters show a significant difference at< 0.05 according to the Duncan’s test. M, Pasting properties of endosperm starch of YK17 and mutants.N, Western blotting of GBSS I in YK17 and mutant rice grains at 10 d after flowering. Actin was used as an internal control.NIP, Nipponbare; YK17, Zhongjiazao 17.

        In conclusion, using an ABE tool, we created novel allelic variations withingene mutants having different AC (3.4%, 16.7% and 25.6%) with varied ECQ properties. Notably, we achieved better ECQ inwith desirable target of moderate AC (16.7%) and higher GC (77 mm), together with the improvement of viscosity, which prevents retrogradation. This study demonstrated a significant strategy for the improvement of AC inriceby introducing novel alleles through genome editing techniques, where most cultivars carry theWxallele and exhibit high AC.

        ACKNOWLEDGEMENTs

        This study was supported by the China National Key Research and Development Program (Grant No. 2020YFE0202300), the Central Public-Interest Scientific Institution Basal Research Fund of China (Grant Nos. Y2020PT07and Y2020YJ09), and the International Science & Technology Innovation Program of Chinese Academy of Agricultural Sciences, China (Grant No. CAAS-ZDRW202109). We are thankful to Prof. Gao Caixia for providing the ABE vector pH-PABE-7-esgRNA.

        SUPPLEMENTAl DATA

        The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

        File S1. Methods.

        Fig. S1.genotype of YK17.

        Fig. S2. Sequencing results of target loci of YK17 and three mutants (,and).

        Fig. S3. Selection of-resistant transgenic plants.

        Fig. S4. Alignment of amino acids ofWxmutants,andconserved in different plant species.

        Table S1. Primers used in this study.

        Table S2. Mutation efficiency in T0generation.

        Table S3. Identification of off-target effects.

        Table S4. Agronomic traits of YK17 and mutants (,and).

        Barman H N, Sheng Z H, Fiaz S, Zhong M, Wu Y W, Cai Y C, Wang W, Jiao G A, Tang S Q, Wei X J, Hu P S. 2019. Generation of a new thermo-sensitive genic male sterile rice line by targeted mutagenesis ofgene through CRISPR/Cas9 system., 19(1): 109.

        Biselli C, Cavalluzzo D, Perrini R, Gianinetti A, Bagnaresi P, Urso S, Orasen G, Desiderio F, Lupotto E, Cattivelli L, Valè G. 2014. Improvement of marker-based predictability of apparent amylosecontent inrice throughallele mining., 7(1): 1.

        Cai X L, Wang Z Y, Xing Y Y, Zhang J L, Hong M M. 1998. Aberrant splicing of intron 1 leads to the heterogeneous 5UTR and decreased expression ofgene in rice cultivars of intermediate amylose content., 14(4): 459–465.

        Fei Y Y, Yang J, Wang F Q, Fan F J, Li W Q, Wang J, Xu Y, Zhu J Y, Zhong W G. 2019. Production of two elite glutinous rice varieties by editinggene., 26(2): 118–124.

        Gaudelli N M, Komor A C, Rees H A, Packer M S, Badran A H, Bryson D I, Liu D R. 2017. Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage., 551: 464–471.

        Huang L C, Sreenivasulu N, Liu Q Q. 2020a.editing: Old meets new., 25(10): 963–966.

        Huang L C, Li Q F, Zhang C Q, Chu R, Gu Z W, Tan H Y, Zhao D S, Fan X L, Liu Q Q. 2020b. Creating novelalleles with fine-tuned amylose levels and improved grain quality in rice by promoter editing using CRISPR/Cas9 system., 18(11): 2164–2166.

        Juliano B O. 1998. Varietal impact on rice quality., 43(4): 207–222.

        Komor A C, Kim Y B, Packer M S, Zuris J A, Liu D R. 2016. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage., 533: 420–424.

        Larkin P D, Park W D. 2003. Association ofgene single nucleotide polymorphisms with starch characteristics in rice (L.)., 12: 335–339.

        Li C, Zong Y, Wang Y P, Jin S, Zhang D B, Song Q N, Zhang R, Gao C X. 2018. Expanded base editing in rice and wheat using a Cas9-adenosine deaminase fusion., 19(1): 59.

        Li H, Li X F, Xu Y, Liu H L, He M L, Tian X J, Wang Z Y, Wu X J, Bu Q Y, Yang J. 2020. High-efficiency reduction of rice amylose content via CRISPR/Cas9-mediated base editing., 27(6): 445?448.

        Liu L L, Ma X D, Liu S J, Zhu C L, Jiang L, Wang Y H, Shen Y, Ren Y, Dong H, Chen L M, Liu X, Zhao Z G, Zhai H Q, Wan J M. 2009. Identification and characterization of a novelallele from a Yunnan rice landrace., 71: 609–626.

        Mikami I, Uwatoko N, Ikeda Y, Yamaguchi J, Hirano H Y, Sujuki Y, Sano Y. 2008. Allelic diversification at thelocus in landraces of Asian rice., 116(7): 979–989.

        Molla K A, Yang Y N. 2019. CRISPR/Cas-mediated base editing: Technical considerations and practical applications., 37(10): 1121–1142.

        Monsur M B, Shao G N, Lv Y S, Ahmad S, Wei X J, Hu P S, Tang S Q. 2020. Base editing: The ever expanding clustered regularly interspaced short palindromic repeats (CRISPR) tool kit for precise genome editing in plants., 11(4): 466.

        Sato H, Suzuki Y, Sakai M, Imbe T. 2002. Molecular characterization of, a novel mutant gene for low-amylose content in endosperm of rice (L.)., 52(2): 131–135.

        Teng B, Zeng R Z, Wang Y C, Liu Z Q, Zhang Z M, Zhu H T, Ding X H, Li W T, Zhang G Q. 2012. Detection of allelic variation at thelocus with single-segment substitution lines in rice (L.)., 30(1): 583–595.

        Tian Z X, Qian Q, Liu Q Q, Yan M X, Liu X F, Yan C J, Liu G F, Gao Z Y, Tang S Z, Zeng D L, Wang Y H, Yu J M, Gu M H, Li J Y. 2009. Allelic diversities in rice starch biosynthesis lead to a diverse array of rice eating and cooking qualities., 106(51): 21760–21765.

        Wanchana S, Toojinda T, Tragoonrung S, Vanavichit A. 2003. Duplicated coding sequence in theallele of tropical glutinous rice (L.)., 165(6): 1193–1199.

        Wang Z Y, Zheng F Q, Shen G Z, Gao J P, Snusted D P, Li M G, Zhang J Z, Hong M M. 1995. Post-transcriptional regulation of the ricegene., 7: 613–622.

        Xu Y, Lin Q P, Li X F, Wang F Q, Chen Z H, Wang J, Li W Q, Fan F J, Tao Y J, Jiang Y J, Wei X D, Zhang R, Zhu Q H, Bu Q Y, Yang J, Gao C X. 2020. Fine-tuning the amylose content of rice by precise base editing of thegene., 19(1): 11–13.

        Yang J, Wang J, Fan F J, Zhu J Y, Chen T, Wang C L, Zheng T Q, Zhang J, Zhong W G, Xu J L. 2013. Development of AS-PCR marker based on a key mutation confirmed by resequencing ofin milky princess and its application insoft rice (L.) breeding., 132(6): 595–603.

        Zeng D C, Liu T L, Ma X L, Wang B, Zheng Z Y, Zhang Y L, Xie X R, Yang B W, Zhao Z, Zhu Q L, Liu Y G. 2020. Quantitative regulation ofexpression by CRISPR/Cas9-based promoter and 5-UTR-intron editing improves grain quality in rice., 18(12): 2385–2387.

        Zhang C Q, Zhu J H, Chen S J, Fan X L, Li Q F, Lu Y, Wang M, Yu H X, Yi C D, Tang S Z, Gu M H, Liu Q Q. 2019.Wx, the ancestral allele of ricegene., 12(8): 1157–1166.

        Zhang C Q, Yang Y, Chen S J, Liu X J, Zhu J H, Lu Y, Li Q F, Fan X L, Tang S Z, Gu M H, Liu Q Q. 2021. A rareallele coordinately improves rice eating and cooking quality and grain transparency.,63(5): 889–901.

        Zhang J S, Zhang H, Botella J R, Zhu J K. 2018. Generation of new glutinous rice by CRISPR/Cas9-targeted mutagenesis of thegene in elite rice varieties., 60(5): 369–375.

        Hu Peisong (hupeisong@caas.cn); Shao Gaoneng (shaogaoneng@caas.cn)

        11 October 2020;

        4 March 2021

        Copyright ? 2021, China National Rice Research Institute. Hosting by Elsevier B V

        This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

        Peer review under responsibility of China National Rice Research Institute

        http://dx.doi.org/10.1016/j.rsci.2021.07.003

        国产精品无码无在线观看| 亚洲av久播在线一区二区| 中文字幕av永久免费在线| 日本二一三区免费在线| 男人的天堂中文字幕熟女人妻| 国产午夜手机精彩视频| 又色又爽又黄还免费毛片96下载| 国产精品v欧美精品v日韩精品 | 午夜国产一区二区三区精品不卡| 婷婷中文字幕综合在线| 国产又黄又爽视频| 久久久久成人精品免费播放| 视频一区二区三区中文字幕狠狠| 在线亚洲精品一区二区三区| 久久久人妻一区二区三区蜜桃d| 亚洲天堂精品成人影院| 性无码一区二区三区在线观看 | 久久精品人人做人人爽| 国产又爽又黄的激情精品视频| 乱人伦视频69| 亚洲女同高清精品一区二区99| 婚外情长久的相处之道 | 少妇厨房愉情理伦bd在线观看| 中文字幕人妻av一区二区| 伊人亚洲综合网色AV另类| 精品少妇后入一区二区三区| 成年人干逼视频水好多| 狠狠躁18三区二区一区| 丰满人妻被黑人猛烈进入| 97精品伊人久久大香线蕉app | 中文字幕日本熟妇少妇| 色婷婷久色国产成人免费| 色噜噜亚洲男人的天堂| 亚洲午夜福利在线视频| 欧美日韩久久久精品a片| 黑人巨大精品欧美在线观看| av成人资源在线播放| av网站免费在线浏览| 亚洲自偷自拍另类第1页| 久久久久久久久蜜桃| 亚洲欧美日韩中文字幕网址|