春季不同程度低水位对四种沉水植物生理的影响
THE PHYSIOLOGICAL EFFECTS OF SPRING LOW WATER LEVEL ON FOUR SUBMERGED MACROPHYTES
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摘要: 为了解不同沉水植物对春季低水位的生理响应, 在2014年春季开展为期3个月的控制实验, 研究不同程度的春季低水位, 包括极低水位(水深18 cm)、较低水位(36 cm)和低水位(54 cm)对3种乡土沉水植物微齿眼子菜、穗花狐尾藻和菹草的最大光化学量子产量(Fv/Fm)、总叶绿素含量和可溶性糖含量的影响, 并与外来种伊乐藻作对比。结果显示, 随着水位的降低, 微齿眼子菜、菹草和伊乐藻Fv/Fm显著升高, 而穗花狐尾藻的Fv/Fm无显著变化; 在3种水位下伊乐藻的Fv/Fm都明显高于其他3种植物。微齿眼子菜和菹草总叶绿素含量也随着水位降低有升高趋势, 而穗花狐尾藻和伊乐藻的总叶绿素含量随水位没有显著变化。所有水位下微齿眼子菜总叶绿素含量最高, 穗花狐尾藻最低, 菹草只在低水位下显著低于伊乐藻。微齿眼子菜、菹草和伊乐藻的可溶性糖含量随着水位的降低而下降, 穗花狐尾藻的可溶性糖含量随着水位的降低有升高趋势。在低水位和较低水位下穗花狐尾藻和菹草的可溶性糖含量分别是所有植物中的最小和最大, 但在极低水位下4种沉水植物的可溶性糖含量无明显差异。以上结果表明, 春季极低水位对微齿眼子菜、伊乐藻和菹草不产生胁迫,但对穗花狐尾藻产生了胁迫; 伊乐藻潜在光合能力强于乡土种, 在春季浅水区具备较强的入侵性。Abstract: To study effects of spring low water level on physiology (such as Fv/Fm, total chlorophyll content and soluble sugar) of different submerged macrophytes, three shallow water levels (18 cm, 36 cm and 54 cm) were treated with three native submerged plants Potamogeton maackianus, Myriophyllum spicatum and P. crispus, and an alien species Elodea nuttallii. The results showed that decreasing water levels increased the Fv/Fm of P. maackianus, P. crispus and E. nuttallii but not on M. spicatum. Fv/Fm of E. nuttallii was significantly higher than that of other three native species at all water levels. The total chlorophyll content of P. maackianus and P. crispus increased with decreasing water levels, while that of M. spicatum and E. nuttallii showed no significant change. The value of chlorophyll content of P. maackianus and M. spicatum was the highest and lowest at all water levels, respectively. The content of total chlorophyll of P. crispus was significant lower than that of E. nuttallii at 54 cm water depth, but other species had no significant difference. The soluble sugar of P. maackianus, P. crispus and E. nuttallii decreased with decreasing water level, while that of M. spicatum increased. The soluble sugar of M. spicatum was the highest and P. crispus was the lowest at both 54 cm and 36 cm depth. However, no significant differences of soluble sugar among four species were found at 18 cm water depth. The results suggest that extremely low water level had stressful impacts on M. spicatum but not on P. maackianus, P. crispus and E. nuttallii, and that the higher potential photosynthesis of E. nuttallii compared with native species would aid its invasive risk in shallow water in spring.
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Keywords:
- Shallow water level /
- Submerged macrophytes /
- Stress /
- Physiology
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TRIM (Tripartite motif)家族蛋白的命名源于这一类蛋白的N端有3个连续的保守结构域: 1个RING (Really Interesting New Gene)、1—2个B-Box及1个CC (Coiled-coil), 合称为RBCC[1]。除了N端的RBCC结构域, 大多数TRIM蛋白还有不同的C端结构域。TRIM家族首先在人中被鉴定命名[2]。此后发现, 脊椎动物的TRIM家族在不同物种中发生了不同程度的扩张。如人有80多个TRIM成员, 但硬骨鱼类除了与人保守的TRIM外, 还存在一类特有的TRIM亚家族, 称为finTRIM(Fish novel TRIM, FTR), 并首先在斑马鱼(Danio rerio)中进行了命名(FTR1-84)[3, 4]。由于finTRIM在不同的鱼类物种中也有特异性扩张, 因此有些finTRIM基因为特定物种的独有基因[4, 5]。
TRIM家族的快速扩张可能与脊椎动物免疫反应的精确调控直接相关[6]。研究表明, 人约50%的 TRIM家族成员参与干扰素(Interferon, IFN)介导的抗病毒免疫反应的调控[7]。IFN是脊椎动物特有的, 在天然抗病毒免疫中发挥重要作用的细胞因子。在病毒感染的细胞中, 病毒复制产生的核酸能被细胞表达的模式识别受体如RLR [Retinoic acid inducible gene-Ⅰ (RIG-Ⅰ)-like receptors]等快速识别, 从而诱导细胞IFN及下游如Mx等称为干扰素刺激基因(IFN stimulated genes)的表达, 抵御病毒入侵[8]。鱼类finTRIM也在IFN抗病毒反应中发挥作用[9]。如斑马鱼FTR36[10]、FTR67[11]和FTR83[12]在鱼类细胞中过量表达时正调控IFN反应。我们实验室最近鉴定的FTRCA1 (FTR Carassius auratus 1)和FTREPC1/2/3/4, 根据目前已知的数据分析表明是物种特异的TRIM基因, 并显现出负调控IFN抗病毒免疫反应的功能[13－15]。
黄颡鱼是我国一种重要的淡水养殖鱼类[16], 每年因各种病害导致巨大的经济损失[17, 18]。目前, 黄颡鱼基因组已经被成功解析[19]。我们在系统分析黄颡鱼的TRIM家族时, 克隆了一个与斑马鱼FTR67同源性高的基因。本文报道对黄颡鱼FTR67基因的克隆和功能初步分析。
1. 材料与方法
1.1 细胞和病毒
3种鱼类细胞系, CAB (Crucian carp C. auratus blastulae embryonic cells)、EPC (Epithelioma papulosum cyprinid)和CO (Grass carp ovary)按本实验常规方法置28℃培养箱培养[20]。SVCV (Spring viraemia of carp virus)在EPC细胞中繁殖扩增并测定病毒滴度。
1.2 黄颡鱼感染、组织RNA提取及RT-PCR
实验用黄颡鱼取自武汉市水产科学研究所(3月龄), 分成两组, 分别腹腔注射1×107 TCID50/mL的SVCV或PBS (每尾300 μL)。在注射0和48h取鳃、心、肝、脾、眼、脑、肠、鳍、头肾、体肾、皮肤和肌肉等12个组织提取总RNA, 用TRUEscript RT MasterMix kit试剂盒(Aidlab公司)合成第一链cDNA。荧光定量PCR实验使用Hieff qPCR SYBR Green Master Mix试剂(YEASEN公司)在Bio-Rad CFX96TM Real-Time system上运行, 每个样本设置3个重复。引物见表 1。
表 1 实验所用引物Table 1. Primers used in this experiment引物名称
Primer序列
Sequence (5′—3′)TfFTR67-F CAGCCTGTCACATCGAGAC TfFTR67-R CCAAAGCTGAGAGATGCCTTAG TfFTR67-F-hm GCCACTGTGCTGGATgccaccATGGCGCAGGCGGGG TfFTR67-R-hm TGGAATTCTGCAGATCTACCGCCGTCTTCTCCG TfIFNpro-F-hm GAGCTCTTACGCGTGgccaccGTCGGGTGATGTTCACA TfIFNpro-R-hm CTCGAGCCCGGGCTAGCATGTTCTCGCTCTCTGCTCG TfFTR67-RT-F CCTTCCTGCAGAGCTACCGG TfFTR67-RT-R GTATTTGACTCCGCATCAGCC TfIFN-RT-F GATCGATAAGGCCAACACAG TfIFN-RT-R CAGTGTCCTGCTGTCCCA TfMx1-RT-F GCGCGAGTCTAAGTGAACAG TfMx1-RT-R AGCTCGAGTGGACATCTTGT Tfactin-RT-F GTCCGTGACATCAAGGAGAAGC Tfactin-RT-R AGGAGGAAGAGGCAGCAGTG EPCIFN-RT-F ATGAAAACTCAAATGTGGACGTA EPCIFN-RT-R GATAGTTTCCACCCATTTCCT EPCMx1-RT-F GGCTGGAGCAGGTGTTGGTATC EPCMx1-RT-R TCCACCAGGTCCGGCTTTGT EPCactin-RT-F CAGATCATGTTTGAGACC EPCactin-RT-R ATTGCCAATGGTGATGAC 1.3 基因克隆及序列分析
根据黄颡鱼基因组中命名为TRIM25-like (GenBank登录号: XP_027021882.1) 的cDNA序列, 在其3′UTR和5′UTR上设计了正向和反向两条引物。以黄颡鱼12个组织的cDNA混样为模板进行PCR扩增。扩增产物序列与登录序列完全匹配, 表明该基因为正常表达基因, 最后命名为TfFTR67。为构建系统进化树, BLAST搜索斑马鱼Danio rerio、金鱼Carassius auratus、草鱼Ctenopharyngodon idella、电鳗Electrophorus electricus、苏氏圆腹?Pangasianodon hypophthalmus、大盖巨脂鲤Colossoma macropomum、虎皮鱼Puntigrus tetrazona、鲤Cyprinus carpio、胖头鱥Pimephales promelas、青鳉Oryzias latipes和红鳍东方鲀Takifugu rubripes等物种基因组, 从每个物种中至少选择一个与TfFTR67最为同源的finTRIM序列。鉴于finTRIM与TRIM16和TRIM25最为同源, 同时搜寻部分TRIM25和TRIM16序列, 一同利用MEGA X软件通过邻近法构建系统进化树。运用在线软件SMART (https://smart.embl-heidelberg.de/)预测蛋白结构、在线软件JASPAR (https://jaspar.genereg.net/analysis)分析基因启动子序列。
1.4 质粒构建
利用同源重组方法, 将TfFTR67的ORF序列插入到真核表达载体pcDNA3.1(-)的EcoR Ⅴ酶切位点构建TfFTR67的真核表达载体。将黄颡鱼IFN基因(GenBank登录号: NW_020847830.1)的启动子序列插入到pGL3-Basic的Nhe Ⅰ酶切位点, 构建荧光素酶基因表达载体(TfIFNpro-luc)。其他表达质粒见本实验室已发表论文[21, 22]。
1.5 细胞转染及荧光素酶活性检测
细胞转染使用Polyethylenimine (PEI, MW25000, 贮存浓度1 μg/μL)并参考本实验室建立的方法进行[20]。细胞接入48孔板过夜, 在第2天加入0.01 μg pRL-TK、0.1 μg启动子质粒、0.1 μg真核表达质粒(3种质粒比例为1﹕10﹕10)的混合液转染。如有需要, 细胞在24h后再次转染poly(I﹕C)(polyinosinic-polycytidylic acid, 1 μg/mL)。细胞用双萤光素酶报告基因系统试剂盒(Promega)裂解, 然后在荧光素酶分析仪(Junior LB 9509)上检测荧光素酶值。每个样品设置3个重复, 每个实验重复3次以上。表达值为每个样品的荧光素酶活性与海肾荧光素酶活性的比值。
1.6 病毒滴度检测
采用微量细胞病变(Cytopathic effect, CPE) 抑制法在EPC细胞中进行。将细胞接入96孔板培养过夜, 去掉培养基, 加入用无血清199培养基10倍梯度稀释的检测样品, 每个稀释度重复8孔, 每孔100 μL。28℃培养5—7d, 待细胞出现明显CPE, 然后加入结晶紫染色。通过TCID50方法计算病毒滴度。
1.7 数据分析
使用GraphPad9.0软件进行差异显著性分析。***表示P<0.001, **表示P<0.01, *表示P<0.05。
2. 结果
2.1 黄颡鱼TfFTR67的克隆鉴定
BLAST分析命名为TRIM25-like (XP_027021882.1)的黄颡鱼基因时, 发现与斑马鱼FTR67同源性高(图 1A), 因此将该基因命名为TfFTR67。此外, TfFTR67与苏氏圆腹?和草鱼的FTR67也具有很高的序列相似性, 但是与含有C端PRY/SPRY结构域的鲫FTRCA1和斑马鱼FTR42相比, 仅在N端具有较高的序列同源性(图 1A)。TfFTR67仅由TRIM蛋白的三联体结构域RING、B-BOX和Coiled-coil组成; 由于缺少C端结构域, TfFTR67蛋白大小明显小于鲫FTRCA1和斑马鱼FTR42(图 1B)。
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图 1 FTR67的氨基酸序列及蛋白结构域组成分析A. 黄颡鱼、斑马鱼、苏氏圆腹𩷶、草鱼FTR67与鲫FTRCA1和斑马鱼FTR42蛋白的多重序列比对; 结构域用黑框表示; (*)和(.和:)分别表示氨基酸的一致性和相似性; B. 黄颡鱼、斑马鱼、苏氏圆腹𩷶、草鱼FTR67与鲫FTRCA1、斑马鱼FTR42的蛋白结构域组成Figure 1. Multiple alignments and domain arrangements of FTR67sA. Multiple alignments of several fish FTR67 proteins and crucian carp-specific FTRCA1 and zebrafish FTR42. The domains of protein are indicated by boxes. The identical (*) and similar (. and :) amino acids are indicated; B. Schematic representation of FTR67 from yellow catfish, striped catfish, zebrafish and goldfish, crucian carp FTRCA1 and zebrafish FTR42 proteins将从斑马鱼等12个物种中搜索的与TfFTR67最为同源的41个序列, 与12个TRIM16和TRIM25序列一起构建系统进化树(图 2)。我们发现所有的TRIM16和TRIM25各聚一支, 而其他聚为一支(finTRIM)。TfFTR67与斑马鱼、金鱼、苏氏圆腹?、草鱼等物种的FTR67形成一个小的进化枝, 同属于finTRIM。尽管在斑马鱼等物种中只找到一个FTR67, 但是在金鱼基因组中发现至少有3个序列与FTR67同源。相反, FTR82、FTR83和FTR84在所有的硬骨鱼类中只有一个直向同源基因(Orthologous gene)。
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图 2 黄颡鱼FTR67的系统进化树分析利用MEGA X软件进行多重序列比对, 在此基础上运用邻近法构建系统进化树Figure 2. The evolutionary relationship of yellow catfish FTR67 with other finTRIM proteinsA neighbor-joining tree is constructed based on analysis of protein sequences using MEGA X program尽管组成FTR67这个进化支的所有序列在公共数据库中命名各异, 但基因共线性分析发现: 3个金鱼FTR67序列分别位于不同的染色体或Scaffold中; 且与黄颡鱼、斑马鱼和苏氏圆腹?FTR67的基因座位相比, 上下游的基因组成非常保守(图 3)。以上结果表明, FTR67在有些鱼类物种如金鱼中发生了基因复制。
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图 3 黄颡鱼、苏氏圆腹?、斑马鱼和金鱼FTR67基因座位的共线性分析通过BLAST分析并结合进化树分析, 三个粗体TRIM25L为金鱼FTR67同源基因, 黑框中的TRIM16L和TRIM25L实际上是FTR87的同源基因, GMRF35L是PIGRL的同源基因Figure 3. Syntenic analyses of FTR67 gene loci from yellow catfish, striped catfish, zebrafish and goldfishBLAST searches combined with phylogenetic tree analyses show that three goldfish TRIM25Ls in bolds are the orthologs of FTR67, the TRIM25L and TRIM16L in boxes are the orthologs of FTR87, and GMRF35L is the ortholog of PIGRL2.2 黄颡鱼FTR67不受病毒诱导表达
RT-PCR结果显示: SVCV感染能显著诱导IFN和Mx1在黄颡鱼组织中的转录表达(图 4A), 但几乎不能诱导TfFTR67的表达 (图 4B)。鉴于被病毒或IFN诱导表达的基因的启动子上一般存在特异的干扰素刺激反应元件[ISRE, 一致序列为 (G/A/T)GAAAN(1-2)GAAA(G/C)(A/T/C)][23, 24], 我们分析了TfFTR67基因的5′侧翼序列, 在距转录起始位点上游的1 kb之内的序列中没有找到可能的ISRE基序(图 4C), 但是能找到如SP1、NF-κB等转录因子的结合基序(图 4D)。因此, TfFTR67是一个组成型表达的黄颡鱼finTRIM基因。
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图 4 TfFTR67在黄颡鱼组织中的表达A. RT-PCR分析TfFTR67、IFN和Mx1基因在SVCV病毒攻毒前后的黄颡鱼12种组织中的转录表达; B. TfFTR67基因的5′侧翼序列; 粗体表示NF-κB结合位点, 斜体表示SP1结合位点, 黑框表示起始密码子, 下划线表示TfFTR67基因的5′非翻译区; C. TfFTR67基因启动子中转录因子结合位点组成; 数字表示相对于TfFTR67基因的转录起始位点的碱基对位置Figure 4. Expression analysis of TfFTR67 in yellow catfish tissues injected with or without SVCVA. RT-qPCR analysis of TfFTR67, IFN and Mx1 transcription in 12 tissues of yellow catfish infected with and without SVCV; B. 5′ flanked sequence of TfFTR67 gene showing the predicated sites specific to different transcription factors, including NF-κB and SP1. The transcription start site is indicated by box; C. Schematic of TfFTR67 promoters showing the location of transcription factor binding sites. Numbers indicate the positions of base pairs relative to the transcription start site of TfFTR672.3 过量表达黄颡鱼FTR67负调控IFN介导的抗病毒反应
我们克隆了TfIFN基因的一段566 bp长的 5′侧翼序列(–620)—(–55), 发现该序列存在2个典型的ISRE元件(图 5A)。用该序列构建TfIFN启动子驱动荧光素酶表达的质粒 (TfIFNpro-luc; 图 5B)。转染poly(I﹕C)及RLR信号通路分子均能诱导TfIFNpro-luc的活性(图 5C), 证明该5′侧翼序列具有启动子活性。
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图 5 过量表达TfFTR67抑制干扰素反应A. 黄颡鱼IFN基因的5′侧翼序列; 黑框表示预测的ISRE基序, 下划线序列用于克隆TfIFN启动子序列构建TfIFNpro-luc质粒的引物, 黑框粗体字表起始密码子; B. TfIFNpro-luc表达质粒示意图; 数字表示相对于转录起始位点碱基对的位置; C. 转染poly(I﹕C)及过量表达RLR信号分子激活黄颡鱼IFN启动子的活性; D和E. 过量表达TfFTR67抑制胞内poly(I﹕C)诱导的TfIFNpro-luc的激活(D)及DrIFNφ1pro-luc的激活(E); F. 过量表达TfFTR67抑制RLR信号分子对DrIFNφ1pro-luc的激活Figure 5. Overexpression of TfFTR67 negatively regulates IFN responseA. 5′ flanked sequences of yellow catfish IFN gene. The putative ISRE motifs are indicated in box, and the underlined highlights the primer for cloning TfIFN promoter DNA to construct TfIFN-pro-luc. The transcription start site is indicated by box and in bold; B. Schematic of TfIFNpro-luc. Numbers indicate the positions of base pairs relative to the transcription start site of TfIFN; C. Yellow catfish IFN promoter is activated by poly (I﹕C) transfection and overexpression of each of RLR signaling molecules; D and E. Overexpression of TfFTR67 inhibit the poly (I﹕C)-triggered activation of TfFTR67pro-luc (D) and DrIFNφ1pro-luc (E); F. Overexpression of TfFTR67 inhibit the activation of DrIFNφ1pro-luc by RLR signaling molecules相同的转染实验还发现: 过量表达TfFTR67抑制poly(I﹕C)对TfIFNpro-luc的激活, 而且具有剂量依赖性; 此外, TfIFNpro-luc的组成型表达也能被过量表达的TfFTR67所抑制(图 5D)。用斑马鱼IFNφ1启动子驱动的荧光素酶质粒(DrIFNφ1pro-luc)进行类似实验, 也获得了相同的结果(图 5E)。以前的研究表明, 转染poly(I﹕C)通过RLR介导的信号途径来诱导IFN的表达[22]。与对照相比, 过量表达TfFTR67同样能够抑制RLR信号分子(RIG-I、MDA5、MAVS、MITA、TBK1、IRF3和IRF7)对鱼类IFN启动子的激活(图 5F)。
2.4 过量表达黄颡鱼FTR67促进SVCV复制
先期过量表达TfFTR67的EPC细胞, 与对照细胞相比, 用SVCV病毒感染后CPE更显著(图 6A), 且细胞上清液中的病毒是对照上清的8倍(图 6B)。在过量表达TfFTR67的细胞中, 内源IFN和Mx1基因的转录受到了显著抑制(图 6C)。以上结果表明, 过量表达TfFTR67可能通过抑制细胞的IFN反应促进病毒的复制。
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图 6 过量表达TfFTR67促进病毒复制在EPC细胞中过量表达TfFTR67, 12h后感染SVCV(5×103 TCID50/mL)。继续培养72h后, 通过结晶紫染色观察细胞CPE(A), 检测细胞上清液中的病毒滴度(B), RT-PCR检测细胞中IFN和Mx1基因的转录(C)Figure 6. Overexpression of TfFRT67 promotes viral replicationEPC cells transfected with TfFTR67 are infected with SVCV (5×103TCID50/mL), followed by crystal violet staining observation (A), viral title detection of supernatants (B), and RT-PC detection of IFN and Mx1 transcripts (C)3. 讨论
TRIM家族首先在人基因中获得系统解析, 并被依次命名[2]。鉴于斑马鱼的基因组相对完整, 因此斑马鱼TRIM家族成员在鱼类中最先获得命名[4]。命名原则: 如果确定与人TRIM存在直向同源的进化关系, 如TRIM16和TRIM25等, 则在斑马鱼中也被命名为TRIM16 和TRIM25; 对于特异于硬骨鱼类的finTRIM家族, 也先期在斑马鱼中依次命名(FTR1-84)[4]。
随着越来越多的鱼类物种被成功解析了基因组, 公共数据库也积累了越来越多的TRIM同源基因。但由于大多没有经过系统的进化分析, 因此, 很多序列在命名上存在着混乱。比如很多finTRIM基因简单以TRIM16L或TRIM25L命名, 以致难以区分这些基因之间的差异。这是因为只要是finTRIM亚家族成员, 就只与finTRIM家族以外的TRIM16和TRIM25最为同源。有些finTRIM基因即使经过了进化树分析, 但是由于在分析时采用的序列数据不足, 因此也可能得出不太正确的结论。如FTR82/83/84在所有鱼类物种中都存在“一一对应”的直向同源基因[4], 但是在公共数据库中, 草鱼FTR84就被命名为FTR99(ARO38113.1; 图 2), 青鳉(XP_004076674.1)和河豚(XP_003970741.1)的FTR84则被简单命名为TRIM16L[13]。
由此可见, 对于物种特异的finTRIM, 如果简单以与斑马鱼的某个FTR同源而命名, 就容易引起误解和混淆。因此我们建议: 鱼类物种特有的finTRIM成员在命名时, 可以加上该物种的种名以便区分。如我们在四倍体鲫(Carassius auratus)中鉴定的鲫特异的finTRIM基因, 就命名为FTRCA1(FTR Carassius auratus 1)[13, 14], 在EPC中鉴定的可能是EPC特异的基因, 分别命名为FTREPC1/2/3/4[15]。TfFTR67的命名是因为与斑马鱼FTR67最同源, 且斑马鱼只有1个FTR67基因。在finTRIM亚家族中, 有些成员在不同鱼类物种中存在“一一对应”的直向同源基因, 有些只在同属、同科或同目的物种中为“一一对应”, 有些却属于物种特有[3, 4]。在金鱼基因组中, FTR67至少有3个成员, 表明鱼类FTR67在有些物种中发生了基因复制、导致基因拷贝数在这些物种中增多, 因此命名可在基因后加上a/b/c区分。虽然我们目前在黄颡鱼基因组中只找到一个FTR67基因, 但因为黄颡鱼基因组现在还不完整, 所以我们还不能确定黄颡鱼只有一个FTR67同源基因。
在功能上, 斑马鱼FTR67被鉴定是一个病毒诱导表达的基因, 且过量表达时能诱导细胞IFN基因的表达、抑制病毒在细胞中的复制[11]。虽然没有黄颡鱼能感染SVCV的报道, 但是我们发现, 黄颡鱼注射SVCV后, 组织中能检测到IFN和Mx的上调表达, 表明注射SVCV能成功诱导黄颡鱼的IFN反应。然而, 对比正常黄颡鱼, SVCV注射的黄颡鱼组织中FTR67的表达几乎不变, 表明黄颡鱼是一个组成型表达的基因。功能研究显示黄颡鱼FTR67负调控细胞IFN基因的表达、促进病毒的复制。金鱼的基因组中存在至少3个FTR67同源基因, 以及TfFTR67与斑马鱼FTR67的表达调控和功能截然相反这些事实表明, 随着鱼类物种的分化, 鱼类FTR67基因在不同物种中的基因拷贝发生了变化, 同时也发生了功能歧化。
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[25] Hussner A, Hofstra D, Jahns P, et al. Response capacity to CO2 depletion rather than temperature and light effects explain the growth success of three alien Hydrocharitaceae compared with native Myriophyllum triphyllum in New Zealand [J]. Aquatic Botany, 2015, 120(part B): 205211
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[29] Ma J, Qin X Y, Zhou X. Study of physiological and biochemical properties of four species of aquatic plants under stress of sewage [J]. Journal of Green Science and Technology, 2013, 5: 5255. [马佳, 覃晓燕, 周希. 四种水生植物在污水胁迫下的生理生化特性研究. 绿色科技, 2013, 5: 5255]
[30] Li J, Lin P, Dong Y, et al. Effect of morphology and physiology of wetland plants on Plateaus at different altitudes [J]. Plant Science Journal, 2013, 31(4): 370377 [李娟, 林萍, 董瑜, 等. 海拔梯度对高原湿地植物形态和生理学效应研究. 植物科学学报, 2013, 31(4): 370377]
[31] Fan S F, Liu C H, Yu D, et al. Differences in leaf nitrogen content photosynthesis, and resource-use efficiency between Eichhornia crassipes and a native plant Monochoria vaginalis in response to altered sediment nutrient levels [J]. Hydrobiologia, 2013, 711(1): 129137
[32] Zhu H, Ma R J. Comparison of photosynthetic characteristics between two hydrophytic invasive plants [J]. Journal of Northwest A F University (Nat. Sci. Ed.), 2010, 38(5): 193198. [朱慧, 马瑞君. 2种水生入侵植物光合特性的比较. 西北农林科技大学学报, 2010, 38(5): 193198]
[33] Zehnsdorf A, Hussner A, Eismann F, et al. Management options of invasive Elodea nuttallii and Elodea canadensis [J]. Limnologica, 2015, 51(March): 110117br [10] Cooke G D. Lake level drawdown as a macrophyte control technique [J]. Journal of the American Water Resources Association, 1980, 16(2): 317322
[34] You W H, Yu D, Xie D, et al. Overwintering survival and regrowth of the invasive plant Eichhornia crassipes are enhanced by experimental warming in winter [J]. Aquatic Biology, 2013, 19(1): 4553
[35] Cui X H, Xiong B H, Pu Y H, et al. Comparative study of regeneration and colonization ability in five submersed macrophytes [J]. Acta Phytoecologica Sinica, 2000, 24(4): 502505. [崔心红, 熊秉红, 蒲云海, 等. 5种沉水植物无性繁殖和定居能力的比较研究. 植物生态学报, 2000, 24(4): 502505]
[36] You H. Study on the ecological adaptiveness of five submersed macrophytes to eutrophic water [D]. Thesis for Master of Science. Nanjing Agricultural University, Nanjing. 2006, pp: 3343. [游灏. 五种沉水植物对富营养化水体的生态适应性研究. 硕士学位论文, 南京农业大学, 南京. 2006, 3343]
[37] Xu W W, Hu W P, Deng J C, et al. Influence of harvesting Potamogeton crispus in a submerged plant community on the growth of submerged aquatic plants and their effects on water quality [J]. Ecology and Environmental Sciences, 2015, 24(7): 12221227 [徐伟伟, 胡维平, 邓建才, 等. 菹草生物量控制对群落中沉水植物生长及水质的影响. 生态环境学报, 2015, 24(7): 12221227]
[38] Xie Y. Study on purification capacity of the aquatic plant communities under different hydrodynamic conditions in water of Taihu Lake [D]. Thesis for Master of Science. Nanjing forestry university, Nanjing. 2012, pp: 1012. [谢宇. 不同水动力下太湖水生植物群落对水体净化能力研究.硕士学位论文, 南京林业大学, 南京. 2012, pp: 1012]
[39] Reto J S, Alaka S, Govindjee. Polyphasic chlorophyll a fluorescence transient in plants and cyanobacteria [J]. Photochemistry and Photobiology, 1995, 61(1): 3242
[40] Arnon D I. Copper enzymes in isolated chloroplasts polyphenoloxidases in Beta vulgaris [J]. Plant Physiology, 1949, 24: 115
[41] Zhang Z L, Qu W J. Experimental Manual of Physiology [M]. Beijing: Higher Education Press. 2004, 127128. [张志良, 瞿伟菁. 植物生理学实验指导. 北京: 高等教育出版社. 2004, 127128]
[42] Warton D I, Hui F K C. The arcsine is asinine: the analysis of proportions in ecology [J]. Ecology, 2011, 92(1): 310
[43] Wang L Z, Wang G X, Ge X G, et al. Influence of different sediment nutrients on growth and photosynthesis fluorescence character isotics of Hydrilla verticillata (L . f) Royle [J]. Acta Ecologica Sinica, 2010, 30(2): 473480. [王立志, 王国祥, 葛绪广, 等. 底质营养盐负荷对轮叶黑藻生长和光合荧光特性的影响. 生态学报, 2010, 30(2): 473480]
[44] Su W H, Zhang G F, Zhang Y S, et al. The photosynthetic characteristics of five submerged aquatic plants [J]. Acta Hydrobiologica Sinica, 2004, 28(4): 391395. [苏文华, 张光飞, 张云孙, 等. 5种沉水植物的光合特征. 水生生物学报, 2004, 28(4): 391395]
[45] Chen K N. Study on biology and ecology of Potamogeton pectinatus L and its application for ecological restoration in Dianchi lake [D]. Thesis for Master of Science. Nanjing Agricultural University, Nanjing. 2002, pp: 6870. [陈开宁. 篦齿眼子菜生物、生态学及其在滇池富营养水体生态修复中的应用研究. 硕士学位论文, 南京农业大学, 南京. 2002, pp: 6870]
[46] Jin P, Hu L W, Jin T X,et al.Responses of the photosynthetic capacity of Elodea nuttallii to three ecological factors[J]. Journal of Hydroecology, 2013, 34(1): 2529. [靳萍, 胡灵卫, 靳同霞,等. 伊乐藻光合能力对三种生态因子的响应,水生态学杂志, 2013, 34(1): 2529]
[47] Chen R Z, Huang S Z, Song S Q, et al. Plant Physiology [M]. Guangzhou: Sun Yat-sen University Press. 1998, 33. [陈润政, 黄上志, 宋松泉, 等. 植物生理学. 广州: 中山大学出版社. 1998, 33]
[48] Hussner A, Hofstra D, Jahns P, et al. Response capacity to CO2 depletion rather than temperature and light effects explain the growth success of three alien Hydrocharitaceae compared with native Myriophyllum triphyllum in New Zealand [J]. Aquatic Botany, 2015, 120(part B): 205211
[49] Li Q. Influence mechanism of environment factors on the growth and development of submerged macrophytes [D]. Thesis for Doctor of Science. Nanjing Agricultural University, Nanjing. 2007, pp: 5063. [李强. 环境因子对沉水植物生长发育的影响机制. 博士学位论文, 南京师范大学, 南京. 2007, pp: 5063]
[50] Simpson D A. Displacement of Elodea canadensis Michx by Elodea nuttallii (Planch.) H. St. John in the British Isles [J]. Watsonia, 1990, 18: 173177
[51] Guo H T, Cao T, Ni L Y. Effects of different nutrient conditions on the growth of a submerged macrophyte, Vallisneria natans, in a mesocosm experiment [J]. Journal of Lake Science, 2008, 20(2): 221227. [郭洪涛, 曹特, 倪乐意. 中等实验规模下不同营养环境对苦草(Vallisneria natans)生长的影响. 湖泊科学, 2008, 20(2): 221227]
[52] Ma J, Qin X Y, Zhou X. Study of physiological and biochemical properties of four species of aquatic plants under stress of sewage [J]. Journal of Green Science and Technology, 2013, 5: 5255. [马佳, 覃晓燕, 周希. 四种水生植物在污水胁迫下的生理生化特性研究. 绿色科技, 2013, 5: 5255]
[53] Li J, Lin P, Dong Y, et al. Effect of morphology and physiology of wetland plants on Plateaus at different altitudes [J]. Plant Science Journal, 2013, 31(4): 370377. [李娟, 林萍, 董瑜, 等. 海拔梯度对高原湿地植物形态和生理学效应研究. 植物科学学报, 2013, 31(4): 370377]
[54] Fan S F, Liu C H, Yu D, et al. Differences in leaf nitrogen content photosynthesis, and resource-use efficiency between Eichhornia crassipes and a native plant Monochoria vaginalis in response to altered sediment nutrient levels [J]. Hydrobiologia, 2013, 711(1): 129137
[55] Zhu H, Ma R J. Comparison of photosynthetic characteristics between two hydrophytic invasive plants [J]. Journal of Northwest A F University (Nat. Sci. Ed.), 2010, 38(5): 193198. [朱慧, 马瑞君. 2种水生入侵植物光合特性的比较. 西北农林科技大学学报, 2010, 38(5): 193198]
[56] Zehnsdorf A, Hussner A, Eismann F, et al. Management options of invasive Elodea nuttallii and Elodea canadensis [J]. Limnologica, 2015, 51(March): 110117
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