利用基因编辑技术改良水稻直链淀粉含量与香味

Improvement of Amylase and Fragrance Levels of Rice by CRISPR/Cas9 System

直链淀粉含量是稻米品质的理化指标之一,理想的稻米直链淀粉含量是13%~18%,有香味的稻米特别受消费者欢迎。本研究利用CRISPR/Cas9系统对Wx、Badh2(fgr)2个基因进行定突变,分别在Wx的5’UTR内含子剪切点和编码区序列(CDS)的第13外显子处设计靶点,Badh2的编码区序列(CDS)的第3和第9外显子处设计靶点,构建2个双靶点CRISPR/Cas9载体,通过遗传转化导入受体‘中早35’中,2个独立的转化事件分别得到14株和13株T0代转化苗,对T0突变情况分析表明,突变频率分别为57.1%,46.2%;对Wx基因编辑后产生的4个无转基因标记的T2代稳定株系进行直链淀粉含量测定,结果分别为12.2%、11.3%、7.4%、4.9%,比未编辑的对照‘中早35’(24.6%)大幅降低;同时对Badh2编辑后产生3个无转基因标记T2代稳定株系进行香气物质2-乙酰-1-吡咯啉(2-AP)含量测定,结果分别为228.16μg/kg,198.31μg/kg,2095.24μg/kg通过口嚼判断这3个株系确实有不同程度的香味,而没有香味的对照‘中早35’为0.000001μg/kg,以上所有株系的农艺性状与对照‘中早35’一致。综合结果表明,利用CRISPR/Cas9系统成功编辑水稻Wx、Badh2基因,获得了稳定遗传、较低直链淀粉含量且带有香味的突变体,为优质稻育种提供了新的种质资源和创建方法。

Amylose content is one of physicochemical indexes of rice quality.The ideal amylose content of rice is13%~18%.Fragrant rice is especially popular with consumers.The Wx and Badh2(fgr)gene were knockouted by CRISPR/Cas9 system and the mutants with suitable amylose and fragrance were obtained.Two target sites were designed at intron splicing sitein 5’UTR and the 13 thexon of the Wx.Two target sites were designed at the 3 thand9 thexon of the Badh2 gene.Two CRISPR/Cas9 vectors were constructed.Then,they were transformed into the receptor named’Zhongzao35’with the Agrobacterium.13 and 14 transgenic plants were produced respectively.The mutation rate of the Wx and Badh2 were 57.1%and 46.2%respectively.The amylose content of 4 homozygous linesof Wx withouttransgenic markerwere 12.2%,11.3%,7.4%and 4.9%respectively in T2 generation,which were lower than’Zhongzao35’(CK,24.6%).The 2-acetyl aroma substances-1-pyrrolinecontent(2-AP)content of 3 homozygous lines of badh2 without transgenic marker were 228.16μg/kg,198.31μg/kg,2095.24μg/kg in T2 gene ration.They have different degrees of fragrance by chewing.The 2-AP of’Zhongzao35’(CK)without fragrance was 0.000001μg/kg.The agronomic traits of all mutants were similar to’Zhongzao35’(CK).The ideal amylose content of rice and fragrance could be generated by CRISPR/Cas9,and new germplasm resources and breeding methods of high-quality rice were provided.