纳米纤维素-铁螯合物矫治梨树缺铁黄化症的转录组分析

Transcriptome Analysis of Nanocellulose-Fe Chelate Correcting Iron-deficiency Chlorosis of Pear

为探究纳米纤维素-铁螯合物矫治梨树缺铁黄化症的分子机制,对水培获得的缺铁黄化杜梨叶片喷施4 mmol/L的FeSO4(处理T1)和纳米纤维素-铁螯合物(纳米纤维素与FeSO4按电荷比1∶3 000螯合,处理T2),以喷施去离子水作对照(CK),处理72 h后,测定叶片活性铁含量、SPAD值和净光合速率,并对叶片进行转录组测序和分析。结果表明,与CK相比,T1和T2处理的叶片活性铁含量分别显著提高110.7%和235.8%,SPAD值显著提高26.1%和61.7%,净光合速率显著提高70.1%和98.5%,T1与T2之间差异也达到显著水平。转录组测序结果显示,相比CK,T1、T2分别有1 033、1 943个差异基因。GO(Gene ontology)功能富集分析显示,在分子功能、细胞组分和生物过程3个部分T2富集的GO项数量均大于T1,主要涉及金属离子固定、氧化还原过程、叶绿体、光合捕光过程等。KEGG(Kyoto encyclopedia of genes and genomes)富集分析发现,与CK相比,T1仅有55个差异表达基因注释到4个通路中,T2有712个差异基因注释到18条通路中,主要参与光合作用-天线蛋白、光合作用、光合组织的碳固定、代谢途径等通路。杜梨铁蛋白家族基因表达量分析显示,T2的4个铁蛋白基因表达量均高于T1。光合作用-天线蛋白和光合作用2个通路中,T1和T2比CK共有58个基因表达量上调,其中56个基因在T2的表达量高于T1。选取8个差异表达基因进行qRT-PCR验证,结果表明,8个差异表达基因的表达模式与RNA-Seq分析结果一致。综合分析,喷施纳米纤维素-铁螯合物能调动更多的基因和代谢通路,使其矫治梨树缺铁黄化症的能力强于FeSO4。

In order to explore the molecular mechanism of nanocellulose-Fe chelate correcting iron-deficiency chlorosis of pear,Pyrus betulifolia leaves with iron-deficiency chlorosis obtained by hydroponics was treated with 4 mmol/L of FeSO4(T1)and nanocellulose-Fe chelate in which nanocellulose and FeSO4 were chelated at a charge ratio of 1∶3 000(T2)by spraying method. Deionized water was served as control(CK). The active iron content,SPAD and net photosynthetic rate of the leaves were determined after 72 h of treatment,and the leaves transcriptome was sequenced and analyzed. The results showed that the active iron contents of T1 and T2 were significantly increased by 110. 7% and 235. 8% compared with the control,the SPAD values were significantly increased by 26. 1% and 61. 7% respectively,the net photosynthetic rates were significantly increased by 70. 1% and 98. 5%,respectively,and there was a significant difference between T1 and T2. The transcriptome sequencing results showed that T1 vs CK and T2 vs CK had 1 033 and 1 943 differentially expressed genes,respectively. GO functional enrichment analysis showed that the number of GO items enriched in T2 was all greater than T1 in molecular function,cell composition and biological process,mainly involving metal ion fixation,oxidation-reduction process,chloroplast,photosynthetic light harvesting. KEGG enrichment analysis found that only 55 differentially expressed genes of T1 were annotated into 4 pathways,and 712 differentially expressed genes of T2 were annotated into 18 pathways compared with the control. These differentially expressed genes were mainly involved in photosynthesis-antenna proteins,photosynthesis,carbon fixation in photosynthetic organisms, and metabolic pathways. According to the analysis of gene expression level of Pyrus betulifolia ferritin gene family,four ferritin genes of T2 were all highly expressed than those of T1. In the pathways of photosynthesis-antenna protein and photosynthesis,the expression levels of 58 genes in T1 and T2 were up-regulated compared with the control,and the expression levels of 56 genes in T2 were higher than in T1. The results of the expression patterns of eight selected differentially expressed genes verified by qRT-PCR were all consistent with the results of RNA-Seq analysis.Comprehensive analysis showed that spraying nanocellulose-Fe chelate could mobilize more genes and metabolic pathways,enhancing its recovery ability from iron-deficiency chlorosis of pear.