【作者】 张涛；

【导师】 于恒秀； 王宝和；

【作者基本信息】 扬州大学 ， 农艺与种业（专业学位）， 2023， 硕士

【摘要】 我国近60%的人口以稻米为主食,提高水稻产量成为育种家们首先要解决的问题。但过去一段时间,水稻新品种的培育过于偏重产量,忽视了稻米品质改良的重要性,随着人民生活质量日益提高,我国的稻米品质已不能完全满足人民的需求。稻米品质性状是由多基因控制,遗传基础复杂,通过常规育种手段进一步改良稻米品质存在难度。新一代基因编辑技术可以快速改良品质性状,提升育种效率。本研究利用CRISPR/Cas9技术获得无抗性选择标记基因的GS9和BADH2双基因编辑株系,并对本实验室前期获得的Wx、GS9、BADH2和SSSII-2单基因或多基因编辑株系通过稻米品质分析、农艺性状考察进一步评价和筛选。主要研究结果如下:1、利用CRISPR/Cas9技术对水稻品系ZZ-16进行GS9和BADH2双基因编辑,获得2个无抗性选择标记基因且农艺性状稳定的长粒香型优良株系;对本实验室前期获得的Wx、GS9、BADH2和SSSII-2单基因或多基因编辑株系后代进行潮霉素抗性基因和目的基因的检测,获得12个无抗性选择标记基因的目的基因突变纯合体。2、Real-time PCR分析结果表明,Wx、GS9、BADH2和SSSII-2基因突变后,大部分转基因株系的目的基因表达量较受体亲本呈显著或极显著降低。3、水稻品种淮稻5号(HD5H)经Wx基因编辑后的株系HDW-1和HDW-2直链淀粉含量(AC)较HD5H极显著降低,接近糯稻(AC<2%)水平,胶稠度(GC)极显著升高,糊化温度(GT)显著降低,说明其米质变软;其淀粉粘滞性谱(Rapid visco-analyser,RVA)特征值显著或极显著降低;米饭食味值较HD5H极显著升高。HD5H经Wx、GS9和BADH2三基因编辑的株系HDGBW-1～3表现出与HDW-1和HDW-2相似的AC、GC、GT和米饭食味值,且籽粒变长,具有香味。4、HD5H和ZZ-16经GS9和BADH2双基因编辑的株系HDGBW-4、HDGBW-5和ZGBW-1、ZGBW-2的粒长较对应受体亲本显著或极显著增加,长宽比极显著增加,具有香味。5、水稻品种抗条武运粳8号(K8)经BADH2和SSSII-2双基因编辑后的株系KBS-1～3的AC较K8显著降低,但高于软米的AC(5%～12%),GC值较K8极显著升高,GT显著降低,说明其米质变软;RVA谱特征值中崩解值较K8极显著升高,峰值粘度、热浆粘度、冷胶粘度和消减值较K8显著降低,米饭食味值较K8提高且具有香味,且其籽粒仍保持亲本的透明度。6、农艺性状统计结果表明,HDW-1、HDW-2和HDGBW-1～3等株系的千粒重较受体亲本显著降低;除部分株系的主穗粒数和结实率较对应受体亲本有变化,大部分株系的其余农艺性状与受体亲本无显著差异。最终,综合农艺性状和稻米品质分析结果,从14个水稻株系中筛选出4个优质稻米新品系:ZGBW-1、HDGBW-1、HDGBW-4 和 KBS-2。更多还原

【Abstract】 Rice is a staple food for nearly 60 percent of the Chinese population.Increasing rice yields has become a priority for breeders and quality improvement was neglected for a long period in the past years.Therefore,rice quality in our country can’t meet people’s demands.Rice quality traits are controlled by multiple genes with complex genetic basis.Therefore,it is not easy to further improve rice quality characters only by conventional breeding methods.For improving the appearance quality,eating and cooking quality(ECQ)of rice,the CRISPR/CAS9 technology was employed to edit Wx,GS9,BADH2 and SSSII-2 genes of 2 rice varieties previously in our lab,which are suitable for cultivated in Jiangsu province.In the present study,new selectable-marker free lines with mutation in both GS9 and BADH2 genes were obtained.For selection of lines with better comprehensive characters,the ECQ and agronomic traits of these lines were further evaluated.The main results are as follows:1.Two selectable-marker free homozygous with mutation in both GS9 and BADH2 genes were obtained from the Japonica line ZZ-16.The genotype of the hygromycin resistance(HPT)gene and the target genes,Wx,GS9,BADH2 and SSSII-2 in the 12 lines obtained previously was confirmed.2.Real-time PCR analysis showed that the expressions of the target genes in most of the lines were reduced accompanying with mutation in the target genes(Wx,GS9,BADH2 and SSSII-2)compared to the wild type(WT).3.The amylose content(AC)of the two lines,HDW-1 and HDW-2 derived from the Japonica variety Huaidao 5(HD5H)with shift mutation in Wx gene was reduced significantly compared to HD5H and close to that of glutinous rice(AC<2%).The gel consistency(GC)of these 2 lines was increased significantly compared to HD5H.Their gelatinization temperature(GT)was reduced significantly.These indicated that they are softer than the WT.Most RVA(Rapid visco-analyser)values of these lines were reduced significantly and taste values were increased significantly.The AC,GC,GT,RVA values and taste values of the three lines,HDGBW-1-3 derived from HD5H with mutation in Wx,GS9 and BADH2 genes were similar to HDW-land HDW-2.And their seeds were longer with fragrance.4.The grain length of HDGBW-4,HDGBW-5 and ZGBW-1,ZGBW-2 derived from HD5H and ZZ-16 respectively with mutation in GS9 and BADH2 genes was increased significantly compared to the corresponding WT.These four lines have fragrance.And the taste values of HDGBW-4 and HDGBW-5 was increased significantly compared to HD5H.5.The AC of the three fragrant lines,KBS-1-3 derived from the Japonica rice Kangtiaowuyunjing 8(K8)with mutation in BADH2 and SSSII-2 genes was reduced significantly compared to K8.And it is higher than that of soft rice(AC 5%～12%).The higher GC and lower GT values indicated softer characters of KBS-1～3 compared to K8.Some RVA values of these three lines were lower than those of K8,including the peak viscosity(PKV),hot paste viscosity(HPV),cool paste viscosity(CPV),and setback viscosity(SBV).Breakdown viscosity(BDV)of KBS-1～3 was increased significantly compared to K8.The taste values of these three lines were higher than that of K8 and their endosperm retained transparency,similar to K8.6.Most agronomic traits of all the lines obtained in present study had no significant change compared to the WT,except for some lines with slight differences in 1000-grain weight,grain number per spike and seed setting rate.Finally,four lines with improved quality and good agronomic traits,ZGBW-1,HDW-1,HDGBW-1,and KBS-2,were selected from the 14 selectable marker-free lines.更多还原