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Cold stress is one of the most common unfavorable environmental factors affecting the growth, development, and survival of plants. The DEAD-box RNA helicases play important roles in all types of processes of RNA metabolism. However, the function of DEAD-box RNA helicase under cold stress is poorly explored in plants, especially in rice. This study reported the identification of a novel rice thermo-sensitive chlorophyll-deficient mutant, tcd33, which displayed an albino phenotype before the four-leaf stage, then withered and eventually died at 20 °C, while wild-type plants exhibited normal green coloration at 32 °C. The tcd33 seedlings also exhibited less chlorophyll contents and severe defects of chloroplast structure under 20 °C condition. Map-based cloning and complementation experiments suggested that TCD33 encodes a chloroplast-located DEAD-box RNA helicase protein. The transcript expression level of TCD33 indicated that the genes related to chlorophyll (Chl) biosynthesis, photosynthesis, and chloroplast development in tcd33 mutants were down-regulated at 20 °C, while the down-regulated genes were nearly recovered to or slightly higher than the WT level at 32 °C. Together, our results suggest that the cold-inducible TCD33 is essential for early chloroplast development and is important for cold-responsive gene regulation and cold tolerance in rice.

In the present study, the transgenic rice “Xiushui 134” with cry1Ac1 was developed by Agrobacterium mediated transformation method. Confirmed by PCR, all the 12 independent transformants were cry1Ac1 -positive plants. Using semi-quantitative reverse transcription PCR (RT-PCR), the cry1Ac1 gene expression could be detected in all independent transformants. Using real time quantitative PCR (qPCR) analysis, we found that 3 out of 12 transformants harbored a single copy cry1Ac1 gene. In order to study the integration site, the flanking sequences of these three transformants were amplified by thermal asymmetric interlaced (TAIL)-PCR. The result showed that the T-DNA of one of them inserted into genes interspace on the rice chromosome 2, about 2.4 kb upstream from the Os02G0539500 (Ch.2: 20858661~20860159) gene, and did not disturb other rice functional genes. This trans- formant was used as primary material to self-cross and segregate, and finally we got the homozygous cry1Ac1 lines. The resistance to leaf folder and agronomic traits of the lines were tested in field at natural conditions. The results showed that the homozygous cry1Ac1 rice plants had strong resistance to leaf folder while main agronomic traits were almost unaffected. These results indicate that we have successfully developed homozygous cry1Ac1 lines with highly resistant to leaf folder but main agronomic traits almost the same as controls.