赤霞珠葡萄黄化突变体生理特性及相关基因分析

赤霞珠葡萄黄化突变体生理特性及相关基因分析

THE STUDY OF PHYSIOLOGY PROPERTY AND RELATED GENE ANALYSIS OF CABERNET SAUVIGNON ALBINO MUTANT

作者：陈迎春　　导师：翟　衡

山东农业大学　　果树学2011届硕士

摘　要

本试验以酿酒葡萄赤霞珠品种(vitis vinifera cv Cabernet Sauvignon)的单侧臂黄化突变体（新梢、叶片及果实均黄化）为材料，通过分析其光合作用、叶绿素、荧光参数、叶片超微结构及13C、15N的吸收调配，初步探明了赤霞珠黄化突变体光合机制及氮素营养代谢运输关系；通过突变株碳水化合物、矿质元素、次生代谢物质及相关酶类测定，基因芯片分析等，为研究突变体生理功能及分子机制提供了理论依据。主要结果如下：

1.　赤霞珠黄化突变体的叶片淡黄、薄脆，叶绿素a、叶绿素b、类胡萝卜素及叶黄素含量均显著低于另侧绿枝叶，黄化成熟叶片分别为绿枝叶片的1.27%、1.10%、7.55%和5.65%；且成熟黄化叶片色素含量低于黄化新叶。

2.　赤霞珠黄化突变体叶片细胞结构存在缺陷。黄化叶内溶物含量少，叶绿体的数目仅为对照株的38.5%；突变体叶绿体形状不规则，无类囊体结构，嗜锇颗粒较多，无淀粉粒；超微结构观察黄化叶片膜系统不完整，核膜、叶绿体膜、线粒体膜均有部分融合现象，胞间连丝增多，没有发现病毒性颗粒和外源性病原体。

3.　突变体叶片的光合性能大幅度下降，2009年7月末测定黄化枝叶片净光合速率Pn和水分利用率WUE仅为绿枝叶的3.66%和8.24%，蒸腾速率Tr为绿枝叶的43.22%，但气孔导度Gs变幅不大；黄化突变体的PSⅡ反应中心活性下降，Fo、Fv、Fv/Fm、Etr、ΦPSⅡ和qP分别为绿枝叶的38.78%、4.09%、55.96%、63.61%、64%和80.39%；黄化叶片的PSII反应中心耗散过剩的激发能力显著增强，NPQ升高了11.50%；黄化枝新叶中叶黄素库(V+A+Z)仅为绿枝叶的9.17%，但与叶片耗散过剩光能的能力大小有关的叶黄素循环效率(A+Z)/(V+A+Z)和绿枝没有显著差异。

赤霞珠突变体绿枝叶光合性能与对照株没有显著差异，但 PSⅡ反应中心活性(Fv)比对照株叶片高31.84%；绿枝叶片PSII反应中心耗散过剩光能的能力(NPQ)也高于对照株叶片14.23%；绿枝叶叶黄素库(V+A+Z)为对照株的1.85倍，但叶黄素循环效率(A+Z)/(V+A+Z)与对照株没显著差异。

4．　13C示踪试验表明黄化下部新梢碳素征调能力强，δ13C值分别是未标记的上部绿梢叶、下部绿梢叶的8.88及45.84倍。突变体叶片对15N的吸收征调能力为绿枝叶的1.3倍；突变体果实对15N的吸收征调能力达到绿枝果实的83%。突变体对氮素营养进行了远距离的调运来满足自身的生长需求，异养特征明显。突变体枝条细弱，髓心比大，枝条不充实，叶片脱落提前，植株有越冬性障碍以致最终死亡。

5.　突变体黄化果穗小，果实单粒重、种子重量均显著低于对照；黄化果实可溶性固形物、可溶性糖、可溶性蛋白的含量分别为对照株的66.67%、60.67%和47.14%；可滴定酸含量显著高于对照，为对照的1.92倍。高效液相色谱法测定赤霞珠果实糖酸种类发现，黄化果实果糖、葡萄糖含量为对照株的65.37%、65.78%，蔗糖含量差异不显著；黄化果实中的酒石酸含量为对照的76.71%，草酸、苹果酸、乳酸、乙酸、柠檬酸含量为对照的3.69、4.26、2.71、2.20和1.84倍，琥珀酸含量没有显著差异。

6.　赤霞珠黄化果实果皮花青素含量极显著低于对照株果实，而果皮中苯丙氨酸解氨酶活性显著高于对照；黄化突变体果皮果肉中总酚含量较高，多酚氧化酶的活性极高，而黄化叶片类黄酮、总酚含量显著低于对照株。

7.　采用基因芯片技术检测了赤霞珠黄化叶片及果实的基因表达谱，在P-value<0.01时，从叶片差异表达基因中筛选到表达发生显著变化的基因2911个，果实差异表达基因775个，两者共同发生显著变化的基因184个。进行功能分类发现，黄化叶片中在植物光合器官的碳代谢、光合作用、卟啉和叶绿素代谢、淀粉蔗糖代谢、激素合成、氨基酸代谢、核苷酸代谢、蛋白质合成、信号转导、细胞膜运输等这些途径中均有基因发生了显著性的差异。

关键词　赤霞珠黄化突变株　光合特性　养分运输　基因芯片

Abstract

Albino side of the Cabernet Sauvignon grapevine was used in this study. We determined the photosynthesis,chlorophyll,chlorophyll fluorescence,ultra microstructure,the absorption and distribution characteristics of 15N-urea and 13C-CO2,and obtained the elementary consequence of photosynthetic mechanism,transportation and distribution of photosynthate in albino clusters. The dates of carbohydrate,mineral element,secondary metabolites and related enzymes,microarray analysis provide theoretical basis for our studying physiological function and molecular mechanism.

1.　The leaves of Cabernet Sauvignon grapevine Albino Mutant were light yellow,thin and fragile. The content of chlorophyll a and chlorophyll b,carotenoid and lutein was significantly lower than that of green leaves，the level of which is only 1.27%,1.10%,7.55% and 5.65% of the green leaves;the pigment content in the mature leaves is lower than that of the young leaves.

2.　The albino mutant cellular has a structure defect. The　intracellular insoluble substance in etiolation leaves dissolved slightly,the number of chloroplast was only 38.5% of the control;the chloroplast had an irregular shape,no thylakoid and starch grain,but a lot of osmiophilic granules;the membrane of etiolation leaves was incomplete,the karyotheca,chloroplast and mitochondrial membrane had a partial inclusion,plasmodesmata number increased,however,no granulosis virus and exogenous pathogens was observed.

3.　The photosynthesis dropped a lot,for example,in July of 2009 photosynthetic rate (Pn) ,efficiency of water application (WUE) and transpiration rate (Tr) were only 3.66%,8.24% and 43.22% of the green branches,respectively;while the stomatal conductance (Gs) changed slightly,;Fo,Fm,Fv/Fm,Etr,ΦPSⅡ and qP was 38.78%,4.09%,55.96%,63.61%,64% and 80.39% of the green branches;while NPQ increased by 11.50%,but there was no significant difference in (A+Z)/(V+A+Z) between the mutant and the green branches.

There is no difference between the green branches in the mutant and in the control. The Fv is 31.84% higher than that of control. The leaves in the same plant is 14.23% higher than that of the control,but no significant difference was observed in (A+Z)/(V+A+Z) between mutant and control.

4.　The mutant is heterotrophic,the leaves of the mutant had a greater ability to absorb and impress the 15N,1.3 times of the green branches. 15N tracer test indicated that the ability of the mutant fruit was 83% of the green branches. The etiolation branches satisfied itself by long-distance transport. The mutant branches were thin and delicate,high proportion of heart centre,which resulted in defoliation of leaf in advance,winter disorder and finally died.

5.　The fruits of the Cabernet Sauvignon Albino Mutant had smaller fruit ear,lighter weight,comparing with the control;the contents of the soluble solid,sugar　and protein was lower than those in the control and were only 66.67%,60.67% and 47.14% of the control. The titrable acidity was 1.92 time of the control. Through HPLC,we found that in the mutant fruits,fructose and glucose were only 65.37% and 65.78% of the control,respectively. Sucrose doesn’t change much. The tartaric acid is 76.71% of the control;while the content of oxalic acid,malic acid,malic acid,acetic acid and citric acid was 3.69,4.26,2.71,2.20 and 1.84 times of the control respectively;however,the succinic acid content didn’t change much.

6.　The fruits of Cabernet Sauvignon Albino Mutant had a light color. And the anthocyanin decreased a lot compares with control,which might be related with PAL activity,while phenol was rich;the etiolation leaves had a lower content of flavonoid and phenol. The PPO activity was much high.

7.　Using gene chip,we detected the gene expression profile of the leaves and fruit in the mutant. Under the situation of P-value<0.01,we got 2911 and 775 genes which changed significantly in the leaves and fruits,respectively. 184 genes changed both in the leaves and fruits. After functional classification,the significantly changed genes in the mutant leaves included the ones in synthesis of plant hormones and proteins,carbon metabolism of photosynthetic organ,photosynthesis,metabolism of porphyrin and chlorophyll,metabolism of starch and sucrose,metabolism of amino group,metabolism of ribonucleotide,signal transduction and membrane transport.

Key words　Cabernet Sauvignon albino mutant　Photosynthetic characteristics　Nutrients transport Gene chip