【作者】 邱丘；

【导师】 杨中艺；

【作者基本信息】 中山大学 ， 环境科学， 2011， 博士

【摘要】 为了应对农田土壤的重金属污染,筛选、培育和利用重金属低积累品种已经成为国际上的研究热点。十字花科芸薹属植物具有较强的重金属吸收能力,容易受到Cd、Pb等重金属的污染。为了降低土壤重金属污染通过食物链威胁人类健康的风险,本研究以芸苔属的叶类蔬菜菜心（Brassica parachinensis）为研究对象,进行菜心镉（Cd）低积累品种的大规模筛选,得到了菜心Cd积累典型品种;随后调查了典型品种在不同土壤和不同采收期的Cd积累稳定性,以及混植和施磷等常见农艺措施降低菜心典型品种Cd积累的效果,并利用差速离心和连续浸提法研究了Cd在菜心典型品种体内的亚细胞分布和化学结合形态,探讨了典型品种间Cd积累的差异和施磷降低菜心Cd积累的主要生化机制,最后分析了菜心典型品种与Cd积累相关基因的单核苷酸多态性,探讨了利用SNP技术对菜心Cd低积累特征进行分子标记的可行性。本研究为明确菜心Cd低积累的生物化学和分子生物学机理提供了理论基础,在实际农业生产应用方面,为通过结合农艺措施与Cd-PSC策略降低菜心Cd污染风险、保障菜心安全生产提供了重要的试验依据。主要研究结果如下:1.通过盆栽试验研究了31个菜心（Brassica parachinensis）品种在对照(0.114 mg kg^-1)、低Cd(0.667 mg kg^-1)、高Cd(1.127 mg kg^-1)污染土壤中的Cd吸收积累情况。在低Cd处理下,全部菜心品种的地上部Cd含量均超出了CAC的限量标准(0.2 mg kg^-1,FW)。在高Cd处理下,品种CLW的Cd含量甚至超出标准高达10倍,说明菜心极易从Cd污染土壤中吸收Cd。方差分析结果表明,菜心地上部Cd含量的品种效应在所有Cd处理下均达到了极显著水平（p < 0.01）,对照、低Cd处理和高Cd处理下的地上部Cd含量分别为0.033 - 0.162 mg kg^-1、0.283 - 1.743 mg kg^-1和0.434 - 2.276 mg kg^-1,品种间最大差异分别为4倍、5.2倍和5.6倍,足以鉴定镉低积累品种,同时地上部Cd含量在各个处理间的相关性都达到了极显著水平（p < 0.01）,说明菜心对Cd的积累能力具有稳定性,是属于可遗传的内在特性。依据上述结果确定了SJ19、49CX、XG49、CH4、LB70和YL80为典型的Cd低积累品种,CLW为典型的Cd高积累品种,用于后续研究。2.通过盆栽试验研究了菜心典型品种对土壤Cd、Pb复合污染的响应。方差分析结果表明,菜心地上部Cd含量的品种效应在所有处理下均达到了极显著水平（p < 0.01）,六个Cd低积累品种的平均Cd含量为0.079 - 0.097 mg kg^-1,而Cd高积累品种为0.242 mg kg^-1,在所有处理下,供试的6个Cd低积累品种的地上部Cd含量均极显著地（p < 0.01）低于Cd高积累品种CLW。7个品种的平均地上部Pb含量范围为0.145 - 0.200 mg kg^-1,排序为CLW > YL80 > XG49 > SJ19 > 49CX > LB70 > CH4,Cd高积累品种CLW在大部分处理下的地上部Pb含量也为最高。方差分析表明,Cd、Pb的交互作用显著（p < 0.01）,土壤Pb在本试验所选取的浓度范围(51.02 - 91.25 mg kg^-1)内明显抑制了菜心对Cd的吸收。根据CAC标准和PSC的定义,在本试验条件下(土壤Cd:0.111 - 0.383 mg kg^-1;土壤Pb:23.50 - 91.25 mg kg^-1),供试的Cd低积累品种均可以看做Cd+Pb-PSCs,而Cd高积累品种CLW为non-Cd+Pb-PSCs。3.探讨了不同土壤和采收期对菜心典型品种Cd积累稳定性的影响。六个供试典型品种在Soil A和Soil B上的地上部Cd含量范围分别为0.026 - 0.080 mg kg^-1和0.452 - 1.349 mg kg^-1。所有品种在Soil A上的Cd含量都要极显著低于Soil B（p < 0.01）,最大相差倍数分别达到了20和13倍,这可能与Soil A的土壤有效P浓度远高于Soil B有关。在两种土壤上,Cd高积累品种CLW和CX4的地上部Cd含量和Cd迁移率都要显著高于（p < 0.05）Cd低积累品种SJ19、CH4、LB70和YL80,说明菜心典型品种的Cd吸收特性在不同土壤上表现稳定。供试菜心品种在采收期T1（第25天）、T2（第40天）和T3（第60天）时的平均地上部Cd含量分别为0.734 mg kg^-1、0.569 mg kg^-1和0.527 mg kg^-1,所有菜心品种的地上部Cd含量均在生长初期最高,随着生长时间的延长而下降,从T1到T2的降幅最为明显。T1、T2、T3期的平均每日Cd累积量分别为0.46μg pot^-1 day^-1、0.87μg pot^-1 day^-1和0.01μg pot^-1 day^-1,可见菜心在生长中期地上部Cd积累的速率最快,是生长初期的1.9倍。不管在哪个采收期,SJ19和CH4的地上部Cd含量都要显著地低于CLW和CX4（p < 0.05）,并且地上部Cd积累量仅为后者的1/3¹/2。可见菜心Cd吸收特性的品种间差异不会随采收期而改变,典型品种的Cd积累特性在不同采收期保持一致。4.以菜心Cd-PSC（SJ19和CH4）和non-Cd-PSC（CLW和CX4）为对象,研究混植效应对菜心典型品种Cd吸收积累的影响。CLW、SJ19、CH4、CX4混植后的地上部Cd含量范围分别为:0.485 - 0.551、0.274 - 0.522、0.233 - 0.423和0.643 - 0.775 mg kg^-1,两Cd低积累品种SJ19和CH4除了与CX4混植后的Cd含量稍微偏高外,其余混植处理下的Cd含量都要显著低于（p < 0.05）Cd高积累品种CLW和CH4。Cd低积累品种与Cd高积累品种CLW混植,两者的地上部Cd含量均无显著变化,而与另一高Cd品种CX4混植,则地上部Cd含量显著上升。两个Cd低积累品种混植则两者的地上部Cd含量与单植相比都会显著下降（p < 0.05）,下降幅度范围分别为24.6%-26.2%和25.8%-28.7%。试验结果表明根际环境在菜心Cd吸收积累方面具有重要作用,选择合适的混植组合可以有效降低菜心受Cd污染的风险。5.以3个Cd低积累品种（Cd-PSC）和2个Cd高积累品种（non-Cd-PSC）为研究对象,研究在不同土壤P水平下,土壤重金属Cd的形态变化以及菜心典型品种对重金属Cd的吸收积累情况。在CK土壤以及CH4和CX4的根际土壤中,可交换态Cd的浓度和比例都随着土壤P水平的升高而下降,与此同时,三种土壤上的铁锰氧化物结合态Cd的浓度和比例随着土壤P水平的升高而增加,推测施磷降低了土壤Cd的生物有效性,减少Cd进入菜心体内的总量。所有供试菜心品种的地上部Cd含量均随土壤P水平的提高呈降低趋势,与P1处理相比,CLW、CX4、SJ19、CH4和LB70在P4处理下的地上部Cd含量分别下降了64%、68%、50%、63%和63%,Cd转运系数分别降低了33.6%、35.4%、23.9%、48.3%和28.0%,可见土壤P水平的提高不仅降低了菜心地上部Cd含量,而且有效抑制了Cd从地下部向地上部的转运。两个Cd高积累品种CLW和CX4的地上部Cd含量始终显著高于（p < 0.05）Cd低积累品种SJ19、CH4和LB70,表明土壤P水平对菜心典型品种的Cd吸收特性的品种间差异并未产生影响。6.利用差速离心和连续浸提法研究了不同土壤P水平下Cd在菜心LB70（Cd-PSC）和CX4（non-Cd-PSC）体内的亚细胞分布和化学结合形态。结果表明LB70细胞壁组分结合的Cd的比例要明显高于CX4,而细胞质组分和细胞器组分的Cd比例却始终低于CX4;LB70体内的NaCl提取态Cd以及残渣态Cd的比例都要高于CX4,然而乙醇提取态和去离子提取态Cd的比例却要低于CX4。因此,LB70细胞壁较强的滞留Cd的能力以及体内Cd较低的移动性可能与其Cd低积累特性相关。土壤P水平的升高明显增加了LB70和CX4与细胞壁组分结合的Cd的比例;两典型品种乙醇提取态Cd的比例随着土壤P浓度的升高而下降,同时离子水提取态Cd和醋酸提取态Cd的比例则随之上升。P可能通过增强菜心体内（特别是地下部）细胞壁组分滞留Cd的能力,以及降低菜心体内无机态Cd的比例等过程,使得Cd在菜心体内的移动性大大下降,从而抑制菜心体内Cd的转运。7.以17个分别具有Cd低积累、中积累和高积累特性的菜心典型品种为研究对象,通过直接测序法,研究了三个已报道的与Cd积累相关的膜蛋白基因（Pcr1、HMA4和NRAMP3）的单核苷酸多态性。在三个基因序列上均发现了丰富的SNP位点。Pcr1、HMA4和NRAMP3基因的SNP发生频率分别为1 SNP/34.4 bp、1 SNP/50.8 bp和1 SNP/34.7 bp,发生转换的位点是颠换的2^-10倍,这说明SNP碱基置换存在偏好性,其主要类型是转换。Pcr1和NRAMP3基因的Indel频率分别为1 Indel/218 bp和1 Indel/84.9 bp,在HMA4基因序列上没有发现Indel,说明HMA4基因比Pcr1和NRAMP3基因保守。根据基因序列的多态性,Pcr1、HMA4和NRAMP3基因分别形成了8、10和10个单倍型（haplotype）,Pcr1基因的haplotype 8包括了5个Cd低积累品种,这个单倍型与菜心的Cd低积累特征基本一致,可以作为一个重金属Cd低量积累的标记用于菜心重金属吸收特性的分子标记辅助选择。Pcr1和HMA4基因的聚类分析表明,菜心Cd低积累品种的基因序列存在较高的相似性,表明其亲缘关系较近。更多还原

【Abstract】 The screening, breeding and exploiting of cultivars that accumulate low heavy metals in edible part has been a hot point to cope with the heavy metal pollution in agriculture soil. It has been reported that plants belonging to cruciferae genus Brassica are high heavy metal accumulator and could be easily polluted by heavy metals such as Cd and Pb. In order to minimize the entry of Cd into the food chain, the present study investigated the Cd accumulation of Chinese flowering cabbage （Brassica parachinensis）, a member of the genus Brassica, for selection of low Cd cultivars. Several tapical cultivars were obtained, and the stability of Cd accumulative characteristics was investigated in different soils and different harvest times. Then, the effets of agricultural approaches such as culture mixture and P application on the Cd accumulation of the typical cultivars were evaluated. The subcellular distribution and chemical speciation of Cd in the typical cultivars were investigated with methods of centrifugation technique and successive extraction, to explain the main biochemical mechanisms of cultivar variation of Cd accumulation and the inhibition effect of P application on Cd accumulation. We also analyzed the single mucleotide polymorphism （SNP） of 17 Chinese flowering cabbage cultivars and investigated the feasibility of identifying low Cd cultivars with molecular biomarkers. This study provided theoretical basis to specific the biochemical and molecular michanisms of the low Cd accumulative characteristic of Chinese flowering cabbage. It also provided the scientifical evidence for the combination of agricultural approaches with Cd-PSC （pollution safe cultivar） stratigy to lower the risk of Cd pollution in Chinese flowering cabbage and ensure the safe production of Chinese flowering cabbage in practical agricultural actions. The main results were as below:1. A pot experiment was conducted to investigate Cd accumulation of 31 Chinese flowering cabbage cultivars under CK (0.114 mg kg^-1), LCd (0.667 mg kg^-1) and HCd (1.127 mg kg^-1) treatments. The shoot Cd concentrations of all cultivars exceeded the maximum limit of CAC for Cd (0.2 mg kg^-1,FW) under the LCd treatment, and the Cd concentration of CLW was even 10 times higher of this limit under HCd treatment. These results indicated Chinese flowering cabbage could readily take up Cd from polluted soils. The variance analysis showed that the variety effect on the shoot Cd concentration of Chinse flowring cabbage was prominent at level of p < 0.01. The shoot Cd concentrations of the tested cultivars under CK, LCd and HCd treatments were 0.033-0.162 mg kg^-1, 0.283^-1.743 mg kg^-1and 0.434-2.276 mg kg^-1. The maximum differences of Cd concentration between the cultivars under the same treatment were 4, 5.2 and 5.6 folds, enough for the seletion of low Cd cultivar. The correlation of shoot Cd concentrations between each treatment were significant at p < 0.01 level, showing the stabllity of Cd accumulation of Chinese flowering cabbage. Six cultivars, SJ19, 49CX, XG49, CH4, LB70 and YL80 were selected as typical low Cd cultivars, and CLW was typical high Cd cultivar.2. The responses of the typical cultivars to the Cd and Pb compound pollution were investigated using a pot experiment. The variety effect on the shoot Cd concentration of Chinese flowering cabbage were significant at p < 0.01 level under all treatments. The average Cd concentrations of six low Cd cultivars were 0.079 - 0.097 mg kg^-1, while that for the high Cd cultivar was 0.242 mg kg^-1. Shoot Cd concentraions of 6 low cultivars were always significantly lower （p < 0.01） than those of the high Cd cultivar, CLW. Shoot Pb concentration of 7 tested cultivars were 0.145 - 0.200 mg kg^-1, in the decreasing order of CLW > YL80 > XG49 > SJ19 > 49CX > LB70 > CH4. The high Cd cultivar accumulated the highest Pb in shoots than other cultivars under most treatments. The variance analysis showed the interaction between Cd and Pb was significant （p < 0.01）. The soil Pb (51.02 - 91.25 mg kg^-1) obviously inhibited the Cd accumulation of Chinese flowering cabbage. According to the definition of CAC and PSC, all the low Cd cultivars could be treated as Cd+Pb-PSCs, and the high Cd cultivar was non-Cd+Pb-PSCs under the soil condition in this study (Cd: 0.111 - 0.383 mg kg^-1; Pb: 23.50 - 91.25 mg kg^-1).3. The present study investigated the effects of different soils and harvest times on the typical cultivars of Chinese flowering cabbage. The shoot Cd concentrations of six typical cultivars in soil A and soil B were 0.026 - 0.080 mg kg^-1 and 0.452 - 1.349 mg kg^-1, respectively. Both the shoot and root Cd concentraions of all cultivars in soil A were significantly （p < 0.01） lower than those in soil B, with the maximum differences being 20 and 13 folds, respectively. It might have relationship with the extremely higher soil available P content in soil A as compared with soil B. The shoot Cd concentrations and transfer ratios of high Cd cultivars, CLW and CX4, were always significantly higher than those of low Cd cultivars, SJ19, CH4, LB70 and YL80, indicating the Cd accumulative characteristics of typical cultivars were stable under different soils. The average shoot Cd concentrations of all tested cultivars for harvest time T1 （25th day）, T2 （40th day） and T3 （60th day） were 0.734 mg kg^-1, 0.569 mg kg^-1 and 0.527 mg kg^-1, respectively. The shoot Cd concentrations of all tested cultivars were highest in the initial growth stage （T1）, decreasing as time goes, with the most obvious decrease from T1 to T2. The average Cd amount absorbed by the tested cultivars each day for the T1, T2 and T3 were 0.46μg pot^-1 day^-1, 0.87μg pot^-1 day^-1and 0.01μg pot^-1 day^-1, respectively, showing the fastest absorption speed of Cd in the middle stage （T2） of growth, being 1.9 folds of initial stage （T1）. The shoot Cd concentrations of low Cd cultivars, SJ19 and CH4, were always significantly lower than those of high Cd cultivars, CLW and CX4. The amount of total Cd in shoots of low Cd cultivars were only 1/3 - 1/2 of those of high Cd cultivars, showing that the harvest time would not change the Cd accumulative characteristics of typical cultivars.4. Taking the Cd-PSCs （SJ19 and CH4） and non-Cd-PSCs （CLW and CX4） as objects for study, the cultivar mixture on the Cd accumulation of typical Chinese flowering cabbage cultivars was investigated. The shoot Cd concentrations of CLW, SJ19, CH4 and CX4 were 0.485 - 0.551、0.274 - 0.522、0.233 - 0.423 and 0.643 - 0.775 mg kg^-1, respectively. The shoot Cd concentrations of SJ19 and CH4 under cultivar mixtures were significantly （p < 0.05） lower than those of CLW and CX4, except that the Cd concentrations of SJ19 and CH4 were a little high when co-cropped with CX4. There was no significant effect of cultivar mixture on shoot Cd concentrations when the low Cd cultivars co-cropped with high Cd cultivar“CLW”, but the shoot Cd concentrations of the low Cd cultivars increased significantly （p < 0.05） when they were co-cropped with another high Cd cultivar“CX4”. When the low Cd cultivar“SJ19”co-cropped with the other low Cd cultivar“CH4”, the shoot Cd concentrations of both culticars significantly （p < 0.05） decreased, with decreses of 24.6%-26.2% and 25.8%-28.7%, respectively. The results showed that rhizosphere plays an important role in the Cd accumulation of Chinese flowering cabbage, and the selection of proper cultivar mixture will effectively decrease the risk of Cd pollution of Chines flowering cabbage.5. This study investigated the soil Cd fractions and the Cd accumulation of three Cd-PSCs and two non-Cd-PSCs under different soil P levels. The concentrations and proportions of exchangeable Cd in the CK soil and rhizosphere soils of CH4 and CX4 decreased with the increasing soil P level, but those of Fe-Mn oxide Cd showed the opposite trend. It assumed that P applying decreased the bioavailability of soil Cd, and thus reduced the total amount of Cd absorbed by Chinese flowering cabbage. The shoot Cd concentrations of all tested cultivars decreased with the increasing of soil P levels. Compared with P1 treatment, the shoot Cd concentrations of CLW, CX4, SJ19, CH4 and LB70 under P4 treatment decreased by 64%, 68%, 50%, 63% and 63%, respectively, and the translocation factors of Cd decreased by 33.6%, 35.4%, 23.9%, 48.3% and 28.0%, respectively. The increase of soil P level not only decreased the shoot Cd concentration of Chinese flowering cabbage, but also effectively inhibited the translocation of Cd from root to shoot. The non-Cd-PSCs always showed higher Cd shoot concentrations than Cd-PSCs, indicating there was no effect of soil P on the Cd accumulative charateristics of the typical cultivars.6. The subcellular distribution and chemical speciation of Cd in LB70 and CX4 under different soil P levels were investigated with the methods of centrifugation technique and successive extraction. The results showed that the proportions of Cd bound to the cell wall fraction were obviously higher in LB70 than in CX4, but the proportions of Cd in the soluble fraction and organelle fraction were always higher in CX4 than in LB70. Compared to CX4, LB70 had lower proportions of Cd in inorganic form and water-soluble from, but higher proportions of Cd in proteins / pectates integrated form, implied that the Cd accumulation in LB70 is associated with the low mobility of Cd in vivo. The increase of soil P level obviously improved the proportions of Cd bound to cell wall fraction and the proportions of Cd in water-soluble form and insoluble Cd-phosphate complexes, but decreased the proportions of Cd in inorganic form. The P application decreased the mobility of Cd in vivo and inhibited the translocation of Cd from roots to shoots through the courses of enhancing the bouding of Cd to cell wall fraction and decreasing proportions of Cd in inorganic form.7. This study investigated the single mucleotide polymorphism of three membrane protein genes which were reported to be associated with Cd accumulation, Pcr1, HMA4 and NRAMP3, in 17 cultivars which were low, middle or high Cd accumulators, respectively. Abundant SNPs were obtained through sequence analysis. The frequencies of SNP in Pcr1, HMA4 and NRAMP3 were 1 SNP/34.4 bp, 1 SNP/50.8 bp and 1 SNP/34.7 bp, respectively. The frequency of transition was 2^-10 folds of transversion, indicating the preference of the base substitution. The frequencies of Indel in Pcr1 and NRAMP3 were 1 Indel/218 bp and 1 Indel/84.9 bp, no Indel was found in HMA4, showing the HMA4 was more conservative than the other two genes. According to the nucleotide polymorphism, the Pcr1, HMA4 and NRAMP3 formed 8, 10 and 10 haplotypes, respectively. The haplotype 8 of Pcr1 included 5 low Cd cultivars, therefore, this haplotype was consistent with low Cd accumulation and could be used as a molecular marker for the assistant selection of low Cd cultivars of Chinese flowering cabbage. The cluster analysis showed that the sequences were similar among the low Cd cultivars which may have close genetic relationship.更多还原