The Gene Response Mechanism Caused by Chinese-fir Fertilization by Transcriptome Technology.. The Gene Response Mechanism Caused by Chinese-fir Fertilization by Transcriptome Technology.

Objective: In order to study the change law of Chinese fir gene expression caused by fertilization, it provides scientific basis for Chinese fir fertilization theory and practice. Method: Using Illumina HiSeq4000 high-throughput sequencing technology, sequencing sequences of four different fertilizer treatment materials (control CK1, nitrogen treatment N1, phosphorous treatment P1, and phosphorous and nitrogen mixture treatment NP1, each with three biological repeats) were obtained, and a series of comparative analysis of the differences were conducted. The experimental material was the 1-year clone cuttings of Chinese fir K62. At the end of 2017year, sand culture experiment was carried out in the plastic greenhouse of research institute of subtropical forestry,CAF. Because it was transcriptome sequencing without reference genome , sequencing reads need to be spliced into de novo, unigene and transcripts are assembled, and then functional annotation, differential expression and other project analysis are conducted. Result: In 12 samples, transcriptome sequencing produced clean reads5.0E+07nt to 7.1E+07nt, and the total splicing length was 724,341,090nt. BLASTX analysis was performed on clean reads in six databases (Nr, Swiss-prot,eggNOG, ,KEGG, Pfam, GO), and 74,288 genes were generated；The Venn diagram reveals the general overview of the gene response of different fertilization treatments: in the N1-VS-CK1 comparison group, NH4NO3 stimulation induced the opening expression of 3,295 genes, and CK1 produced 2,944 genes resistant to nitrogen; Similar results were obtained for other comparison groups. Chinese fir fertilization turns on and up-regulates the expression of some genes while shutting down or down regulating some genes of expression; Analysis of gene differential expression revealed that :720 differentially expressed genes were screened out in the N1-VS-CK1 comparison group, and 3,694 genes were found to be silenced by nitrogen application. Similar results were obtained in the other five groups: a number of differentially expressed genes were screened out, and a number of genes were silenced by nitrogen and phosphorus fertilizer; The results of the heatmap clustering graph of the six comparison groups were revealed that the genes between the same treatment and the same comparison group are basically distributed in blocks: either high expression or low expression, and vice versa. The gene distribution between different treatments in the same comparison group was complementary. To fertilize Chinese fir, it is better to mix fertilizer with different types of fertilizers; The GO enrichment and functional classification has three major categories, namely, biological process BP, cell component CC and molecular function MF, with 50 small categories; Fertilization, whether single fertilizer or multiple fertilizers, has the greatest impact on the GO terms of Chinese fir: chloroplasts, cytoskeleton, cell membrane, nucleus and cytoplasm. secondly molecular function, protein binding and biological process are affected. These results revealed that nitrogen and phosphorus can promote the absorption and metabolism of themselves and each other; KEGG enrichment analysis was performed in 6 comparison groups, and the most significant first 5 Pathway KEGG terms were selected in each comparison group, the comprehensive analysis results showed that after fertilizing Chinese fir, the most significant KEGG term was photosynthesis, nitrogen metabolism, carbon fixation and carbon metabolism, plant hormone signal transduction, plant-pathogen interaction, phenylpropanoid biosynthesis, pentose-phosphate pathway, porphyrin and chlorophyll metabolism, arginine biosynthesis and ect. Based on the above studies, the significantly up-regulated or down-regulated genes in important terms obtained by GO enrichment and classification and KEEG enrichment can be studied, and the results can guide the diagnosis of Chinese fir fertilization. Conclusion: Illumina HiSeq4000 transcriptome sequencing is a quick and cheap way to study the molecular mechanisms of non-model plants, including coniferous trees with large genomes. Chinese fir fertilization can not only turn on some genes, but also turn off some genes. The growth and development of Chinese fir will be regulated in a way that is favorable for its adaptation to the environment, competitive resources and growth and development. Overall design: Four different fertilizer treatment materials (control CK1, nitrogen treatment N1, phosphorous treatment P1, and phosphorous and nitrogen mixture treatment NP1, each with three biological repeats) were obtained,using Illumina HiSeq4000.

研究目的：为解析施肥诱导杉木基因表达的变化规律，为杉木施肥的理论研究与生产实践提供科学支撑。
方法：采用Illumina HiSeq4000高通量测序（high-throughput sequencing）技术，对4组不同施肥处理材料（对照组CK1、氮肥处理组N1、磷肥处理组P1、氮磷混合肥处理组NP1，每组设置3次生物学重复（biological repeats））进行测序，获得测序序列并开展一系列差异比较分析。实验材料为杉木K62的1年生无性系插条，于2017年末在中国林科院亚热带林业研究所塑料温室开展沙培试验。由于本研究为无参考基因组（reference genome）的转录组测序（transcriptome sequencing），需对测序reads进行从头组装（de novo），组装得到unigene与转录本，随后开展功能注释（functional annotation）、差异表达分析（differential expression analysis）等项目研究。
结果：12个样本的转录组测序共获得clean reads 5.0×10^7 nt至7.1×10^7 nt，总拼接长度达724341090 nt。对clean reads在Nr、Swiss-prot、eggNOG、KEGG、Pfam、GO共6个数据库中进行BLASTX分析，共注释得到74288个基因。维恩图揭示了不同施肥处理的基因响应概况：在N1-VS-CK1比较组中，硝酸铵（NH₄NO₃）刺激诱导3295个基因表达激活，对照组CK1中存在2944个氮响应抗性基因；其余比较组得到相似结果。杉木施肥可激活并上调部分基因的表达，同时抑制或下调部分基因的表达水平。差异表达分析结果显示：N1-VS-CK1比较组中共筛选得到720个差异表达基因，其中3694个基因在施氮后表达沉默。其余5个比较组得到相似结果：均筛选得到一定数量的差异表达基因，且存在部分因氮、磷肥施用而表达沉默的基因。6个比较组的热图聚类分析结果表明：同一处理及同一比较组内的基因基本呈模块分布，要么高表达要么低表达，反之亦然；同一比较组内不同处理间的基因表达分布呈互补模式。上述结果提示，杉木施肥采用多类型肥料配施效果更佳。GO富集分析与功能分类包含生物过程（BP）、细胞组分（CC）、分子功能（MF）3大类，共50个小类。无论施用单一肥料还是复合肥料，对杉木GO条目影响最为显著的类别为叶绿体、细胞骨架、细胞膜、细胞核与细胞质；其次为分子功能、蛋白质结合及生物过程相关条目。上述结果表明，氮与磷可相互促进彼此的吸收与代谢过程。对6个比较组开展KEGG富集分析，每组选取富集程度最高的前5条KEGG通路条目。综合分析结果显示：杉木施肥后显著富集的KEGG通路主要包括光合作用、氮代谢、碳固定与碳代谢、植物激素信号转导、植物-病原体互作、苯丙烷生物合成、磷酸戊糖途径、卟啉与叶绿素代谢、精氨酸生物合成等。基于上述研究，可进一步对GO富集分类与KEGG富集分析得到的重要通路中显著上调或下调的基因开展深入研究，其结果可为杉木施肥诊断提供指导。
研究结论：Illumina HiSeq4000转录组测序是研究基因组较大的非模式植物（包括针叶树）分子机制的快速且经济的技术手段。杉木施肥不仅可激活部分基因的表达，同时可抑制部分基因的表达，进而通过调控杉木生长发育，使其更适应环境、竞争资源并促进自身生长。
整体实验设计：采用Illumina HiSeq4000测序技术，对4组不同施肥处理材料（对照组CK1、氮肥处理组N1、磷肥处理组P1、氮磷混合肥处理组NP1，每组设置3次生物学重复）进行转录组测序。