关键词: 水稻, OsRRK1, 胞质类受体激酶, 叶片卷曲, 叶绿素含量

Abstract:

【Objective】OsRRK1 (RoP-Interacting receptor-like kinase 1) is a member of the cytoplasmic receptor kinases RLCK VI family. In this study, the leaf morphology of OE-OsRRK1 transgenic rice was observed to clarify the role of OsRRK1 in the leaf development of rice. 【Method】Primers were designed based on target sequences published in the rice genome database to construct OsRRK1 overexpression vector, then transformed to Japonica variety Hejiang 19 mediated by Agrobacterium tumefaciens. Positive plants were identified by PCR analysis. The copy number of inserted exogenous genes in transgenic plants was identified by Southern blot hybridization. The expression level of transgenic plants at RNA level was confirmed by Northern blot hybridization and qRT-PCR. T₂ generation homozygote plants at heading stage was selected to determine the leaf rolling index (LRI). Paraffin sections and aniline blue staining were used to observe the changes of rice bulliform cells after cross-cutting, and Image J software was used to calculate the area of the bubble cells. Chlorophyll content in plant leaves was measured by chlorophyll tester.【Result】44 positive transgenic plants were obtained and 17 lines were randomly selected for Southern blot analysis. Among the 17 lines, 8 lines were single-copy insertion and 9 lines were multi-copy insertion. 2 multi-copy lines (OE-1 and OE-4) and 5 single-copy lines (OE-5, OE-21, OE-22, OE-24 and OE-25) were randomly selected for subsequent analysis. qRT-PCR analysis revealed that OsRRK1 was overexpressed with different degree in different transgenic lines. Among them, OE-1, OE-4 and OE-25 lines with the highest OsRRK1 expression level, while OE-5 lines with the lowest and OE-21, 22 and 24 lines with the middle. Three single copy transgenic lines OE-5, OE-22 and OE-25 and WT control were selected for Northern blot analysis. The results were in consistent with the results of qRT-PCR results. Leaf rolling index of the seven selected lines and WT control revealed that the degree of leaf curl was positive correlated with the expression level of OsRRK1, with the higher the expression level, the higher the degree of rolling. Paraffin section and staining revealed the number and area of bulliform cells in the flag leaves of OE-OsRRK1 transgenic plants changed obviously compared to WT. OE-OsRRK1 transgenic plants showed less bulliform cell number than those in the control whose average is 4.6. OE-OsRRK1 transgenic plants with curlier leaf, with the more severe bulliform cell degeneration, less number, smaller area. Chlorophyll content determination revealed that the OE-22, OE-24 and OE-25 lines have higher chlorophyll content than those of the WT rice leaves.【Conclusion】The overexpression of OsRRK1affect the number and size of bulliform cells in rice leaves, which cause leaf rolling, and the degree of leaf rolling is positively correlated with the expression level of OsRRK1. The overexpression of OsRRK1 gene will lead to the increase of chlorophyll content in rice.

Key words: Oryza sativa, OsRRK1, cytoplasmic receptor-like kinase, leaf rolling, chlorophyll content