利用CRISPR/CAS9技术编辑水稻香味基因Badh2

【目的】香稻作为一类特殊的水稻群体,以其清香可口的品质特性备受消费者的欢迎。到目前为止,水稻中的香味主要受第8染色体上编码甜菜碱醛脱氢酶基因Badh2控制。【方法】通过CRISPR/CAS9技术对中花11的香味基因Badh2进行编辑。【结果】获得转基因T_0代植株并对其所衍生的T_1代20个单株进行了鉴定分析,获得了一个剔除了载体骨架且第1外显子上插入一个碱基T的突变体材料。该材料中Badh2 RNA水平显著下调;利用GC-MS技术测定野生型及突变体材料籽粒2-乙酰-1-吡咯啉含量,结果表明突变体材料中的香味物质显著增加;此外,我们还对野生型及T_2代香型植株水稻产量及稻米蒸煮食味品质进行了考查及测定分析,发现除分蘖数及结实率呈现出显著差异外(P<0.05),其余各项指标在两组材料间都无显著差异。【结论】通过CRISPR/CAS9技术成功地对水稻香味基因进行了编辑,可为香稻育种提供丰富的理论指导,加快香稻的育种进程。

控制水稻中胚轴伸长的QTL定位

水稻中胚轴能够为幼苗破土提供动力,培育长中胚轴水稻品种有助于水稻直播技术的推广,因此,研究水稻中胚轴伸长具有重要的理论和现实意义。为分析调控水稻中胚轴伸长的遗传基础,以Asominori和IR24重组自交系(RIL)群体为材料,结合其连锁图谱,对2017年杭州收获种子的中胚轴长度性状进行QTL定位。结果表明,在Asominori/IR24重组自交系群体中共检测到3个控制中胚轴伸长的QTL位点,分别位于第2、第3和第7条染色体上,LOD值在2.34~3.41之间,单个QTL对表型贡献率在7.25%~11.07%之间。同时用Asominori遗传背景的IR24染色体片段代换系进行验证,qML2对应的代换家系CSSL12的中胚轴与Asominori相比显著伸长,qML7对应的代换家系CSSL37的中胚轴与Asominori相比显著缩短,从而验证了qML2和qML7位点的存在。与先前研究比较,qML3和qML7在不同群体不同环境下稳定表达。同时利用重组自交系群体对2018年杭州大田环境株高性状进行QTL定位,共检测到2个QTL位点,均与中胚轴QTL位点不重合,说明控制中胚轴伸长与控制株高有着不同的遗传基础。

Editing of Rice Isoamylase Gene ISA1 Provides Insights into Its Function in Starch Formation

Isoamylase 1(ISA1) is an isoamylase-type debranching enzyme which plays a predominant role in amylopectin synthesis. In this study, the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated endonuclease 9(CRISPR/Cas9) system was used to edit ISA1 gene in rice via Agrobacterium-mediated transformation. We identified 36 genetic edited lines from 55 T_0 transgenic events, and classified the mutation forms into 7 types. Of those, two homozygous mutants, cr-isa1-1(type 1, with an adenine insertion) and cr-isa1-2(type 3, with a cytosine deletion) were selected for further analysis. Seed sizes of both cr-isa1-1 and cr-isa1-2 were affected, and the two mutants also displayed a shrunken endosperm with significantly lower grain weight. Electron microscopy analysis showed that abnormal starch granules and amyloplasts were found in cr-isa1-1 and cr-isa1-2 endosperm cells. The contents of total starch, amylose and amylopectin in the endosperm of the cr-isa1 mutants were significantly reduced, whereas sugar content and starch gel consistency were observably increased compared to the wild-type. The gelatinization temperature and starch chain length distributions of the cr-isa1 mutants were also altered. Moreover, transcript levels of most starch synthesis-related genes were significantly lower in cr-isa1 mutants. In conclusion, the results indicated that gene edition of ISA1 affected starch synthesis and endosperm development, and brought potential implications for rice quality breeding.

Differences of Physicochemical Properties Between Chalky and Translucent Parts of Rice Grains

Rice grain chalkiness is an important characteristic,but the difference between chalky and translucent parts in grains is still unclear.Here,we investigated the differences of flour made from the chalky or translucent part of rice grains in three indica and three japonica rice varieties.The chalky flour had significantly lower amylose and protein contents and looser starch granule morphology,and starches in the chalky flour had higher relative crystallinity,higher short chain content but lower long chain content than those in the translucent flour.The water states,determined with nuclear magnetic resonance,differed between the chalky and translucent flour after soaking,cooking and retrograding,and the chalky flour had more bound and free water but less constructural water than the translucent flour.Mostly,the chalky flour had lower viscosity and shorter gel consistency,but higher onset temperature and gelatinization enthalpy than the translucent flour.The results indicated that starch granule morphology would be more indicative than other attributions on pasting and gelatinization properties of chalky and translucent parts of rice grains.

Editing of Rice Endosperm Plastidial Phosphorylase Gene OsPho1 Advances Its Function in Starch Synthesis

OsPho1 in Zhonghua 11(ZH11)was edited using the clustered regularly interspaced short palindromic repeatsassociated endonuclease 9(CRISPR/Cas9)system.Two homozygous T1 mutants(cr-pho1-34 and cr-pho1-37)displayed a chalky endosperm with a white core,which significantly decreased 1000-grain weight.In addition,many rounded starch granules and abnormal amyloplasts were present in the central region of mutant endosperm cells with increased amylose and lipid contents,decreased total protein content,and altered physicochemical properties of starch.The OsPho1 protein is localized in chloroplasts,and quantitative real-time PCR(qRT-PCR)andβ-glucuronidase(GUS)staining indicated that OsPho1 was highly expressed in seeds at 5 d after fertilization(DAF).OsPho1 mutations displayed close relationships with plastidial phosphoglucomutase and ADPGlc pyrophosphorylase based onα-D-glucose-1P at different temperatures.Moreover,the expressions of starch metabolismrelated genes were also altered in the mutant,and the overexpression of OsPho1 may cause grain chalkiness.

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

Amylose content(AC)is the key determinant of eating and cooking quality(ECQ)of rice.The major Wxa allele of granule-bound starch synthase I(GBSS I)in indica rice produces higher AC,making rice hard and dry after cooking.Recent work has improved ECQ of japonica rice via clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)or cytosine base editing(CBE)techniques.

NRL3 Interacts with OsK4 to Regulate Heading Date in Rice

NRL3 is essential for the growth and development of rice leaves.In this study,we found that the loss function of NRL3 also delayed heading date under natural long daylight and short daylight conditions.The yeast two-hybrid and the bimolecular fluorescence complementation proved that NRL3interacts with OsK4,a Snf1-related kinase.OsK4 localized to the nucleus and expressed in various rice tissues.The rhythmic expression pattern of Os K4 was similar to NRL3 under long daylight and short daylight conditions.Knock-out mutants of Os K4 exhibited early heading under long daylight conditions,indicating that it acts as a negative regulator of heading date in rice.Interestingly,the OsK4 mutant under the nrl3 mutant background rescued the late heading phenotype of nrl3 under long daylight conditions,suggesting that Os K4 functions downstream of NRL3.Moreover,both NRL3 and Os K4 controlled heading date through regulating the expression of Hd3a and RFT1 genes.These findings shed light on the heading date regulation in rice and provide a sound theoretical base to improve regional adaptability of rice.

一个调控水稻叶绿素合成基因wl-1的精细定位

为改良优异两系不育系株1S (Z1S)的株型特征,利用甲基磺酸乙酯(EMS)诱变技术处理株1S (Z1S),得到一个苗期为白条纹的突变体,命名为wl-1。以该突变体与粳稻品种日本晴杂交,得F1种子,进而获得自交F2群体。遗传分析表明wl-1受单个隐性基因控制。利用图位克隆技术将wl-1初定位于水稻第3号染色体上RM15851和RM15880两个标记之间。进一步扩大遗传定位群体,最终将wl-1精细定位于标记inedl3和indel4之间的122 kb区域内。生物信息学分析表明该区域有12个ORF。对这12个ORF编码区逐个测序分析表明,其第10个ORF (LOC-03g52170),编码4-羟基-3-甲基丁-2-烯基二磷酸还原酶的第2个外显子上的第165位碱基处存在1个碱基A替换为T的突变。基因表达分析表明,在幼苗期与野生型株1S相比,突变体中LOC-03g52170的表达量显著低于相应的野生型;同时突变体中叶绿素合成相关基因、光合作用相关基因以及叶绿体发育相关基因的表达量发生了显著变化。对叶色基因wl-1 (LOC-03g52170)的挖掘,有利于进一步阐释叶绿体和叶绿素合成的生物学机理,同时为水稻高光效育种提供理论和技术支撑。

稻米质构特性影响因素的研究进展

随着生活水平的提高,人们对稻米品质尤其是食味品质的要求也随之提高。米饭的质构特性被认为是米饭食味品质评价中最关键的指标。本研究围绕大米组成成分、不同蒸煮处理方法、以及加工储存条件等影响米饭质构特性的主要因素进行了总结,并讨论了质构仪测定米饭质构特性过程中不同参数设置对测定结果的影响。此外,还介绍了目前稻米质构特性遗传研究情况并讨论了今后稻米质构研究中有待加强的方面,旨在为稻米质构特性评价及优质食味水稻品种选育提供参考。