Expression of lncRNA SVR and adjacent SAUR genes in rice during seed germination under low temperature
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摘要:目的
长链非编码RNA(Long non-coding RNA，lncRNA)长度大于200 nt，一般不具有编码蛋白质功能。探究低温下lncRNA对邻近SAUR基因表达的影响，以期为研究水稻种子低温萌发的能力提供理论依据。
方法lncRNA SVR由华南农业大学国家植物航天育种工程技术研究中心前期研究筛选，可以响应低温胁迫，通过生物信息学方法分析lncRNA SVR的二级结构并寻找lncRNA SVR内部的冷胁迫基序，通过qRT-PCR分析lncRNA SVR和SAUR基因的表达特性。
结果lncRNA SVR序列中存在高度相似的冷胁迫响应基序，且在茎环连接处。表达特性分析表明，在种子萌发过程中低温胁迫会持续降低lncRNA SVR的表达，邻近的多个SAUR基因在低温胁迫下的表达量明显高于在常温下的表达量，表明lncRNA SVR的邻近SAUR基因一定程度上能够和lncRNA SVR一样响应低温胁迫。表达相关性分析表明，在低温萌发中lncRNA SVR与这些SAUR基因的表达均呈负相关关系，lncRNA SVR与OsSAUR55的表达呈显著负相关关系。进一步分析种子低温萌发中SAUR基因在lncRNA SVR敲除系中的表达情况，结果表明，在敲除系中，lncRNA SVR的下降幅度低于其在野生型中的下降幅度，OsSAUR41、OsSAUR53、OsSAUR54、OsSAUR55表达量的上升幅度均显著低于其在野生型中的上升幅度。
结论lncRNA SVR可能负调控OsSAUR55的表达，进而响应低温胁迫。
Abstract:ObjectiveLong non-coding RNA (lncRNA) is longer than 200 nt and generally does not code proteins. To explore the effect of lncRNA on the expression of neighbouring SAUR genes under low temperature, and provide a theoretical basis for studying the ability of rice seeds to germinate at low temperature.
MethodlncRNA SVR was investigated in response to cold stress in previous research of National Engineering Research Center of Plant Space Breeding, South China Agricultural University. The secondary structure of lncRNA SVR was analysed by bioinformatics, and the cold stress motifs within lncRNA SVR was searched. The expression characteristics of lncRNA SVR and SAUR genes were analysed by qRT-PCR.
ResultIn this study, highly similar cold stress response motifs were found in the sequence of lncRNA SVR and at the stem-loop junction. Expression characterization showed that cold stress during seed germination continuously decreased the expression of lncRNA SVR, and the expression of some neighbouring SAUR genes was significantly higher under cold stress than that at room temperature, indicating that the neighbouring SAUR genes could respond to cold stress to some extent as well as lncRNA SVR. Expression correlation analysis showed that the expression relationship between lncRNA SVR and these SAUR genes in low temperature germination were all negatively correlated, and the expression of lncRNA SVR and OsSAUR55 showed a significant negative correlation. Further analysis of SAUR gene expression in low temperature seed germination in the lncRNA SVR knockout lines showed that the decrease of lncRNA SVR in the knockout lines was lower than that in the wild type, and the increase in expression of OsSAUR41, OsSAUR53, OsSAUR54 and OsSAUR55 was significantly lower than that in the wild type.
ConclusionlncRNA SVR is able to negatively regulate the expression of OsSAUR55, further respond to low temperature stress.
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图 1 lncRNA SVR序列中存在的冷胁迫响应基序
红色标识为与冷胁迫响应基序高度相似序列的位置，黄色标识为与冷胁迫响应基序存在高度相似性的序列
Figure 1. Cold stress response motif in lncRNA SVR
The positions of highly similar sequences to the cold stress response motifs are marked in red, and the sequences showing high similarity to the cold stress response motifs are marked in yellow
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图 2 lncRNA SVR在常温和低温萌发中表达的动态变化
Figure 2. Dynamic changes of lncRNA SVR expression in seed germination at room and low temperatures
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图 3 SAUR基因簇在常温和低温的表达分析
Figure 3. Expression analysis of SAUR gene cluster at room and low temperature
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图 4 lncRNA SVR和SAUR基因簇表达的相关性分析
Figure 4. Expression correlation analysis of lncRNA SVR and SAUR genes
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图 6 不同SAUR基因在lncRNA SVR转基因材料中的表达分析
Figure 6. Expression analysis of different SAUR genes in lncRNA SVR transgenic materials
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图 7 不同SAUR基因在lncRNA SVR转基因材料中的表达变化
“*”“**”分别表示在P<0.05和P<0.01水平差异显著(t检验)
Figure 7. Expression changes of different SAUR genes in lncRNA SVR transgenic materials
“*” and “**” indicate significant differences at P<0.05和P<0.01 levels respectively (t test)
表 1 本研究所使用的引物
Table 1 qRT-PCR primers used in this study
名称 Name ID/基因 Gene 序列(5′→3′) Sequence lncRNA SVR(用于时空表达
For temporal and spatial expression)MSTRG.182510.4 F: TCTGTGCGATGAGACTCAAGGAG R: AGCCCATAACGTCATGCTTGC lncRNA SVR(用于基因敲除材料
For gene knocked out material)MSTRG.182510.4 F: TTCACAAGAGACTGTCCCATCGTA R: TGCTACGAGAACCACGTTTGC OsActin Os03g0718100 F: GAATGCTAAGCCAAGAGGAG R: AATCACAAGTGAGAACCACAG OsSAUR39 Os09g0545300 F: CCATGATCAATCCTAAGAGGCT R: TGATCTTTTCCTCACCTGTGAA OsSAUR40 Os09g0545400 F: ATCTTGATTTGTGTAGCATCGC R: GACAACAACCTGACTAGACTGA OsSAUR41 Os09g0545700 F: ACCTCTCCCCATGTTCTGGT R: ACCCTGAAGGCGACTATTGC OsSAUR44 Os09g0547033 F: TTGTGTCTGCTTAGGAGAGATG R: TGTACAAACAGCAAGTTGATGG OsSAUR45 Os09g0546100 F: CAGCTGAAACCAAGATCAACAA R: GTGCAGCACTCTTCATTTTCTT OsSAUR46 Os09g0546200 F: GAGACTGGTTCAATTGGCAAAG R: GAGCATTCTTCAGTTTCTTGGG OsSAUR51 Os09g0546700 F: GATGGCCTACCTCGCTGCTG R: GCATTTGGTTGGAGACAAGGC OsSAUR53 Os09g0546900 F: GAGTATGTCCTGTGCTTGCTTA R: CAAGATTTCACAATGGTGCTCA OsSAUR54 Os09g0547000 F: CCACAGCGTATGTCGCAGA R: GATCGCCGTGTTGAGGTACA OsSAUR55 Os09g0547100 F: AGAAATGGCGAAGGATGGCA R: AACCTTGGGCTTCTTCCGTC 
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