Construction Of Normalized Long-Fragment RNAi Library Of Maize Leaf, Root And Kernel Tissues, And Establishment Of RoGFP1-Based Biosensor For Cellular Redox Changes In Maize

Maize is the largest crop in terms of yield and cultivated area in China. It is not only the major source of food and feed, but is also the important industrial raw material. Therefore, it plays a pivital role in ensuring national food and economic security. Although maize yield has increased in recent years, the average yield per unit area is only 60% of that produced in USA, suggesting that there is a great potential for yield increase per unit area in China. Plant architecture, inflorescence/ear architecture, and stress resistance comprise major yield traits of maize, and are thus the major limiting factors for yield increase. We have been focusing on ROS-mediated signaling network regulating stress resistance and kernel development, and have identified the related genes in the lab. To further identify more ROS-regulated genes involved in stress-resistance and developmental genes in maize, genome-wide normalized long-fragment RNAi libraries of maize leaf, root and kernel tissues and roGFP-based sensing system for cellular redox changes were constructed in this study by using mothods such as phi29-mediated rolling amplification, transient expression in maize mesophyll protoplasts, Agrobacterium-mediated transformation of maize embryos, fluorescence intensity ratio analysis, and real-time quantitative PCR.Results obtained in this study are as follows:◇ Using the leaves of plants at three-leaf stage treated with PEG6000 as the material, a normalized maize leaf cDNA library was constructed, which is composed of 1.25×10 clones with 1.5 kb average size of insert fragments. By using rolling amplification, the normalized cDNA library was converted to a RNAi library, which contains 3×106 clones with 3kb average size of insert fragments. Random sequencing and annotated function analysis of 1000 RNAi clones revealed that cDNA sequences evenly distributed on chromosome with biological functions, metabolic and signaling transduction pathway. The gene silencing effect of ihRNAs in the library was confirmed in tranformed maize mesophyll protoplasts by using real-time quantitative PCR.◇ Using the roots of plants at three-leaf stage treated with PEG6000 as the material, a normalized maize root cDNA library was constructed, which is composed of 1.25 x 106 clones with 1.5 kb average size of insert fragments. By using rolling amplification, the normalized cDNA library was converted to a RNAi library, which contains 3×104 clones with 3 kb average size of insert fragments. Random sequencing and annotated function analysis of 500 RNAi clones revealed that cDNA sequences evenly distributed on chromosome with biological functions and involved in metabolism including TCA cycle pathway.◇ Using kernel as the material, a normalized maize kernel cDNA library was constructed with 1.0 kb average size of insert fragments. By using rolling amplification, the normalized cDNA library was converted to a RNAi library with 2 kb average size of insert fragments.◇ In maize mesophyll protoplasts, a roGFPl-based sensing system responding to redox changes caused by the addition of H2O2 and DTT, APX perturbation and synergistic factors was constructed. Meanwhile, we made an overall phylogenetic analysis of Z. mays APX family, implying that APX family in Z. mays plays an important role in detoxifying ROS.10 of the APX genes responded to ABA/stress induction with different expression patterns, indicating a role in modulating ROS and redox state under stress in maize and therefore, were used as marker genes in the establishment of the roGFPl-based sensing system.These results provided genome-wide nomorlized RNAi libraries for further construction of maize genome-wide RNAi mutant populations, maize genetic improvement, a roGFP-based screening system for identifying the stress-resistance and developmental genes which are mediated by ROS signaling.