Using CRISPR/Cas9 gene editing technology to verify the functions and applications of genes related to rice yield, insect-resistance, disease-resistance and aromatic traits

• Main Authors: Yu-Ting Feng, 馮鈺庭
• Other Authors: Liang-Jwu Chen
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/49n35n

碩士 === 國立中興大學 === 分子生物學研究所 === 107 === Rice (Oryza sativa) is one of the most important crops worldwide and feeds more than half of the world’s population. In the face of continuously increasing population and reduced arable land, how to further improve the grain yield of rice is a major concern of scientists and breeders. Random natural mutations or artificial mutagenesis are very inefficient in producing large numbers of mutant plants or mutating specific genes. CRISPR/Cas9 gene editing technology can mutate to target regions and produce high-density mutant populations. This study used the CRISPR/Cas9 to edit the grain yield-related genes DEP1, Gn1a, qTGW3 (which have been reported to function as regulators of panicle density, grain number and size respectively) in rice cultivar Tainung 71. Preliminary analysis of yield-related traits in T0 plants indicated that obtaining similar mutations in each target genes would have the opportunity to affect the morphological traits so as to increase yield in new rice cultivars. Rice production suffers from insect pests. Brown planthopper is one of the most serious pests in rice production, which causes up to 10-100% yield loss annually. To increase Tainung 71 BPH-resistance, several BPH-resistance related genes, such as CYP71A1, GNS5, Bph29 that to regulate plant hormone or reduce callose degradation to interrupt BPH feeding were modified by CRISPR/Cas9. The editing efficiency of T0 plants is 0% (for CYP71A1) or more than 90% (for GNS5 promoter editing) indicating the editing efficiency of CRISPR/Cas9 vary depending on the gene or editing position. For future applications, screening for transgene-free rice line with its edited gene to become homozygous is required in each following generations. Finally, insect resistance test to gene-edited line is required to verify its BPH-resistant characteristic to reach the goal of obtaining a BPH-resistant Tainung 71 cultivar. Furthermore, rice blast-resistance mutant SA0169 and unique aroma mutant SA0420 are derived from rice blast-susceptible and non-aromatic Tainung 67 rice variety by sodium azide mutagenesis. The gene verification for both SA0169 and SA0420 has not been confirmed. It is presumed to be caused by rice blast-resistance gene Nbs1/Pi9 and Nbs2/Pi9 or aroma-related gene badh2 mutation. This study used overexpression or CRISPR/Cas9 editing strategies to perform gene function verification and to create rice blast-resistance with aroma traits rice cultivar as well. The results showed that Nbs1/Pi9 and Nbs2/Pi9 sequence contained unexpected mutations, which may be caused by random mutation of sodium azide or un-homogenization of the plants. The genes of Nbs2/Pi9 and badh2 in transgenic plants were not edited, which may be caused by not-well sgRNA design and poor editing efficiency.Using CRISPR/Cas9 to obtain a specific gene-edited mutant is a rapid and effective method. If these gene-edited plants have stable genotype with improved traits, and the mutation of rice blast resistance and aromatic trait genes can be verified, their future application via gene editing in rice breeding can be assured.