中国科学院海洋研究所机构知识库

     近年来，化肥和农药的大规模使用造成了土壤肥力下降，环境污染加重等问题，这给农业的可持续发展带来了挑战；与此同时，人们对粮食安全及食品品质问题越来越重视。壳寡糖是一种自然界中广泛存在的线性寡糖，由于其具有多种生物活性，壳寡糖已应用到诸多行业中去。在农业上，大量研究表明，壳寡糖能促进植物的生长发育。并且，壳寡糖的聚合度与其生物活性密切相关，根据生物活性的不同，其构效关系也存在差异。但以往的研究大都是以包含多种聚合度的壳寡糖混合物为材料研究其促生长活性。因此，很难知道具体是哪些聚合度的壳寡糖片段在其促生长活性方面发挥了主要作用。此外，以往对壳寡糖促进植物生长发育的研究大都局限于植物对壳寡糖的形态学和生理学响应，而壳寡糖对植物代谢的具体调控机制还不清楚。为了使壳寡糖能更好地应用到农业中去，本论文从下面两个方面开展研究工作：一是对壳寡糖聚合度与其促生长活性间的构效关系进行研究；二是通过代谢组学和转录组学技术，研究了小麦幼苗对壳寡糖的代谢响应情况以及相应的代谢调控机制。具体研究结果如下：
1. 9 种单一聚合度或窄聚合度（壳二糖、壳三糖、壳四糖、壳五糖、壳六糖、壳七糖、壳八糖、DP 8-10 和 DP 10-12）的壳寡糖处理小麦幼苗后，我们对各处理组中小麦幼苗的根长、苗高、干重、鲜重以及光合作用和叶绿素荧光相关的参数进行了测定。结果表明，壳寡糖的促生长活性与其聚合度密切相关，并且聚合度大于3是壳寡糖发挥促生长活性的结构基础。与其他寡糖相比，壳五糖、壳六糖、壳七糖、壳八糖和DP 8-10对小麦幼苗生长参数以及光合作用相关参数的促进作用更好，其中，壳七糖的活性最好。在壳七糖处理组中，小麦幼苗的根长、干重和鲜重明显增加，可溶性糖、可溶性蛋白和叶绿素含量分别增加了59.4%、22.0%和20.3%。各聚合度壳寡糖对光合作用和叶绿素荧光相关参数的影响与其对生长参数的影响相一致。在壳七糖处理组中，小麦幼苗叶片中的净光合速率（Pn），光系统Ⅱ的潜在活性（Fv/Fo），光化学淬灭系数（qP）和可变荧光下降比值（Rfd）分别提高了35.2%、11.0%、18.6%和14.7%，而非荧光淬灭系数（NPQ）则降低了48.6%，说明壳寡糖能促进小麦幼苗的光合作用，提高光能利用率。
2. 根据壳寡糖的聚合度与其促生长活性间的构效关系的研究结果，我们选取活性较好的壳六糖、壳七糖和壳八糖，通过代谢组学的方法（GC-TOF-MS）进一步研究了小麦幼苗对壳寡糖的代谢响应情况。代谢组学结果表明，壳六糖、壳七糖和壳八糖均能诱导小麦幼苗基础代谢过程（光合碳固定、糖酵解、TCA循环和基本氮代谢等）中有机酸、糖类和氨基酸含量的变化。壳六糖、壳七糖和壳八糖处理后分别诱导小麦幼苗产生了29、55和48个差异代谢物。这一结果表明，与壳六糖相比，壳七糖和壳八糖对小麦幼苗代谢组学的影响更大。通过对差异代谢物进行KEGG分析发现，壳七糖处理组中的差异代谢物主要参与了小麦幼苗中的光合碳固定、TCA循环、丙酮酸代谢以及丙氨酸、天冬氨酸和谷氨酸代谢；壳八糖主要参与了光合碳固定、淀粉和蔗糖代谢以及半乳糖代谢；然而，壳六糖则主要激活了植物的果糖和甘露糖代谢。因此，壳七糖在激活植物碳氮代谢方面的活性相对更好。
3. 为了进一步对代谢组学的结果进行验证，我们选取壳七糖进一步从代谢物水平，相关酶活及基因表达水平层面研究了壳寡糖对小麦幼苗碳氮代谢的影响。结果表明，壳七糖可以促进小麦幼苗光反应的进行，提高小麦幼苗的蔗糖含量。蔗糖磷酸合成酶（SPS）和果糖-1,6-二磷酸酶（FBPase）是蔗糖合成过程中的两个关键酶，它们的酶活和基因表达水平均明显提高。在TCA循环中，磷酸烯醇式丙酮酸羧化酶（PEPC）和丙酮酸脱氢酶（PDH）的酶活及其基因表达水平的提高可能进一步导致了中间代谢物草酰乙酸和苹果酸含量的增加。此外，壳七糖还能明显促进植物的氮还原和氮同化。在壳七糖处理组中，谷氨酸和天冬氨酸的含量明显高于对照组，小麦幼苗中与这些差异氨基酸合成相关的酶，包括硝酸还原酶、谷氨酸合成酶、谷氨酰胺合成酶、谷氨酸脱氢酶和谷草转氨酶等的活性也明显升高。因此，壳七糖处理可以综合调控植物的碳氮代谢。
4. 为了进一步研究壳寡糖对植物的代谢调控机制，本论文继续通过转录组学的方法，分析了壳七糖处理后，小麦幼苗叶片中的mRNAs和microRNAs（miRNAs）应答情况。我们共鉴定到了400个差异表达的mRNAs，包括268个上调mRNAs和132个下调mRNAs。通过对差异表达mRNAs进行GO富集分析和KEGG富集分析，我们发现壳寡糖可以调控与植物光合作用、基础碳氮代谢、防御应答以及转录因子等相关基因的差异表达。此外，miRNAs也参与了壳寡糖对植物生长发育相关代谢的调控。与对照相比，壳七糖处理组中小RNA（sRNAs）的长度分布在18 ~ 30 nt间有明显变化。我们鉴定出了87个已知miRNAs和21个新miRNAs，其中包含56个差异表达miRNAs。其中，壳七糖诱导miRNA156，miRNA159a，miRNA164和miRNA171a的表达水平显著下调，而miR167c，miRNA319和miRNA1127的表达水平明显上调。

    Conventional crop production is increasingly being challenged by various problems such as decreased soil fertility and pollution due to the use of hazardous chemical pesticides and fertilizers at a global scale. In the same time, there has been mass awareness of quality and safety of food production. This situation escalates public concerns regarding the use of eco-friendly biostimulants which contribute to not only improve plant growth and development but also produce organic greens. Chitooligosacchrides (COS) is natural linear oligosaccharide and exhibits numerous interesting physicochemical and biological properties, which make it suitable for use in many fields. In agriculture, numerous studies had reported that COS can be used to promote the plant growth. Moreover, it is reported that the bioactivity of COS is closely related to its degree of polymerization (DP). Depending on different bioactivities, the function–structure relationship between DP of COS and its bioactivities could be different. However, most of previous studies were conducted using heterogeneous COS with different DP. On the basis of those prior studies, it was hard to confirm which COS with well-defined DP played a leading role in  promoting plant growth. In addition, as for the bioactivity of COS on promoting plant growth, previous studies mostly focused on the apparent effects of COS on the plant physiology and growth characteristics. There are few reports about the metabolic response mechanism of plant to COS. In order to provide a fundamental guidance for future large-scale application of chitosan in agricultural, this thesis was performed to investigate the size effect of COS on its growth-promoting effect and COS-induced metabolic regulation on plant via metabonomics and transcriptomics technologies. Results were displayed as follows:
1. After treated with nine COSs with well-defined DP, including seven single COSs (chitobiose to chitooctaose) and two COS fractions with narrow degrees of polymerization (DPs) (DP8-10, DP10-12), we determined the size effects of COSs on the shoot length, root length, dry weight, fresh weight, and parameters related to photosynthesis and chlorophyll fluorescence of wheat seedlings in all treatment groups. The results showed that the activities of COS on plant growth were closely related to their DPs, and DP > 3 was necessary to insure a significant promotion effect on the growth and photosynthesis. (GlcN)₅, (GlcN)₆, (GlcN)₇, (GlcN)₈ and DP 8-10 seemed to be more effective than other COS fragments, and (GlcN)₇ exhibited the optimal activity. (GlcN)₇ could significantly increase the root length, dry weight and fresh weight. The contents of soluble sugar, soluble protein and chlorophyll in (GlcN)₇ treatment group was improved by 59.4%, 22.0% and 20.3%, respectively. The size effect of COS on photosynthesis was in accordance with its effect on the growth parameters of wheat seedlings. The values of net photosynthetic rate (Pn), PSⅡ potential photochemical efficiency (Fv/Fo), photochemical quenching coefficient (qP) and variable chlorophyll fluorescence decrease ratio (Rfd) in (GlcN)₇ treatment group were improved up to 35.2%, 11.0%, 18.6% and 14.7%, separately, while nonphotochemical quenching coefficient (NPQ) was decreased 48.6%, which resulted in the enhancement of photosynthesis and the promotion of light utilization efficiency of wheat seedlings.
2. According to the analysis of structure-fuctional relationship between the DP of COS and its growth-promoting effect, the most effective COS fragments [(GlcN)₆, (GlcN)₇ and (GlcN)₈] were applied in our experiments to performed an integrative analysis of metabolite profiles with GC-TOF-MS in COSs-treated wheat seedlings. Metabolite profiling revealed that three chitosan fragments all could induce significant difference of organic acids, sugars and amino acids involved in photosynthetic carbon metabolism, glycolysis, tricarboxylic acid (TCA) cycle and central nitrogen metabolism in leaves of wheat seedlings. (GlcN)₆, (GlcN)₇ and (GlcN)₈ induced 29, 55 and 48 differentially changed metabolites, respectively, which suggested that (GlcN)₇ and (GlcN)₈ were much more effective than (GlcN)₆ in regulating plant metabolism. According to the KEGG analysis, the differentially changed metabolites in (GlcN)₇ treatment were mostly involved in carbon fixation in photosynthetic organisms, TCA cycle, pyruvate metabolism, and alanine, aspartate and glutamate metabolism in wheat seedlings. (GlcN)₈ mainly induced the carbon fixation in photosynthetic organisms, starch and sucrose metabolism and galactose metabolism. However, (GlcN)₆ mainly activated the fructose and manose metabolism. It seemed that (GlcN)₇ was more effective in activating the metabolic response of wheat seedlings relative to primary C and N metabolism.
3. In order to further validate the results of metabolite profiles and give a more comprehensive understanding of growth-promotion effect of COS, we therefore selected (GlcN)₇ as a representative to investigate its impacts on primary C and N metabolism at metabolites, enzyme activities and transcript levels. Our results showed that (GlcN)₇ could promote the light reaction in photosynthesis of wheat seedlings. A higher accumulation of sucrose content was also observed after (GlcN)₇ treatment, accompanied by an increase in sucrose phosphate synthase (SPS) and fructose 1, 6-2 phosphatase (FBPase) activities as well as their up-regulation of relative expression level. Several metabolites associated with TCA cycle, including oxaloacetate and malate, were also improved along with an elevation of phosphoenolpyruvate carboxylase (PEPC) and pyruvate dehydrogenase (PDH) activities and their transcription expression levels. On the other hand, (GlcN)₇ could also enhance the N reduction and N assimilation. Glutamate, aspartate and some other amino acids were higher in (GlcN)₇-treated plants, accompanied by the activation of key enzymes of N reduction and N assimilation, including nitrate reductase (NR), glutamate synthase (GOGAT), glutamine synthetase (GS), glutamate dehydrogenase (GDH) and glutamic oxalacetic transaminase (AAT). Together, these results suggested COS could induce a pleiotropic modulation of carbon and nitrogen metabolism in wheat seedlings.
4. In order to further reveal the metabolic response mechanism induced by COS on plant and how COS regulate the metabolic pathwsays, we simultaneously investigated mRNAs and microRNAs (miRNAs) expression profiles of wheat seedlings in response to (GlcN)₇. We identified 400 chitosan-responsive differentially expressed genes, including 268 up-regulated and 132 down-regulated mRNAs. According to GO analysis and KEGG analysis, we found (GlcN)₇ could regulate the genes expression involved in photosynthesis, primary carbon and nitrogen metabolism, defense responses and transcription factors. Moreover, miRNAs also participate in (GlcN)₇-mediated metabolic regulation on plant growth. Compared with the control, the length distribution of small RNAs (sRNAs) was significantly changed in (GlcN)₇ library. We identified 87 known and 21 novel miRNAs, among which 56 miRNAs were induced or repressed by chitosan heptamer. Specialy, miRNA156, miRNA159a, miRNA164, miRNA171a were significantly down-reguated, while miR167c, miRNA319 and miRNA1127 were up-regulated obviously.