中国青藏高原青稞抗穗发芽资源评价与α-淀粉酶基因的克隆及表达

穗发芽（PHS，preharvest sprouting）是影响禾本科作物生产的重要的灾害之一。收获时期如遇潮湿天气容易导致穗发芽发生。发生穗发芽的种子内部水解酶（主要是α-淀粉酶）活性急剧升高，胚乳贮藏物质开始降解，造成作物产量和品质严重降低。因此，选育低穗发芽风险的品种是当前作物育种工作中面临的重要任务。 青稞(Hordeum vulgare ssp. vulgare)主要分布于青藏高原，自古以来就是青藏高原人民的主要粮食。近年来，由于青稞丰富的营养成分和特有的保健品质、在燃料工业中的潜力以及在啤酒酿造工业中的利用前景，在发达国家日趋受到重视，掀起综合研究利用的热潮。我国拥有占全世界2/3 以上的青稞资源，具有发展青稞产业的得天独厚的条件。然而，由于青稞收获期间恰逢青藏高原雨季来临，常有穗发芽灾害发生，使青稞生产损失巨大。目前对青稞穗发芽研究很少，适用于育种的穗发芽抗性材料相对缺乏，不能很好的满足青稞穗发芽抗性育种的需要。本研究以青藏高原青稞为材料，对其穗发芽抗性的评价指标和体系进行构建，同时筛选青稞抗穗发芽品种并对其抗性进行评价，还利用分子生物学手段对青稞穗发芽抗性的分子机理进行了初步探讨。主要研究结果如下： 1. 本试验以来自于我国青藏高原地区的青稞为材料，对休眠性测定的温度范围进行探讨，并对各种穗发芽抗性测定方法的对青稞的适用性进行评测。通过探讨温度对13 个不同基因型的青稞籽粒发芽和休眠性表达的影响，对筛选青稞抗穗发芽资源的温度条件进行探索，并初步分析了其休眠性表达的机理。在10，15，20，25，30℃的黑暗条件下，选用新收获的13 个青稞品种为材料进行籽粒发芽实验，以发芽指数（GI）评价其休眠性。结果发现，不同品种对温度敏感性不同，其中温度不敏感品种，在各温度条件下均表现很低的休眠性；而温度敏感品种，其休眠性表达受低温抑制，受高温诱导。15℃至25℃是进行青稞休眠性鉴定的较适宜的温度范围。通过对供试材料发芽后的α-淀粉酶活性，发现温度对青稞种子的休眠性表达的影响至少在一定程度上表现在对α-淀粉酶活性的调控上。随后，对分别在马尔康和成都进行种植的34 份青稞穗发芽指数（SI），穗发芽率（SR），籽粒发芽指数（GI）和α-淀粉酶活性（AA）进行了测定和分析，发现它们均受基因型×栽培地点的极显著影响，且四个参数之间具有一定相关性。GI 参数由于其变异系数较低，在不同栽培地点稳定性好，且操作简便，是较可靠和理想的穗发芽评价参数。SI 参数可作为辅助，区别籽粒休眠性相似的材料（基因型）或全面评价材料（基因型）的穗发芽抗性特征。AA 参数稳定性较差，并且检测方法复杂，因此不建议在育种及大量材料筛选和评价时使用。此外，青稞穗发芽抗性受环境影响较大，评价时应考虑到尽可能多的抗性影响因素及其在不同栽培条件下的变异。 2. 对来自青藏高原的青稞穗发芽抗性特征及其与其它农艺性状间的关系进行研究。通过测定穗发芽指数（SI）、籽粒发芽指数（GI）和α-淀粉酶活性（AA），表明113 份青稞材料的穗发芽抗性具有显著差异。SI、GI 和AA 参数的变幅分别为1.00~8.86、0.01~0.97 和0.00~2.76，其均值分别为4.72、0.63 和1.22。根据SI 参数，六个基因型，包括‘XQ9-5’，‘XQ33-9’，‘XQ37-5’，‘XQ42-9’，‘XQ45-7’和‘JCL’被鉴定为抗性品种。综合SI、GI 和AA 参数，可以发现青稞的穗发芽抗性机制包含颖壳等穗部结构的抗性和种子自身的抗性（即种子休眠性），且供试材料中未发现较强的胚休眠品种，除‘XQ45-7’外，所有品种在发芽第四天均能检测出α-淀粉酶活性。穗部结构和种子休眠的抗性机制因基因型不同而不同，在穗发芽抗性中可单独作用或共同作用。农家品种和西藏群体分别比栽培品种和四川群体的穗发芽抗性强，而在不同籽粒颜色的青稞中未发现明显差异。相关性检验发现，青稞的穗发芽抗性，主要是种子休眠性，与百粒重、开花期、成熟期、穗长、芒长和剑叶长呈显著负相关关系，与株高相关性不显著。农艺性状可以作为穗发芽抗性材料选育中的辅助指标。本试验为青稞穗发芽抗性育种研究提供了必要的理论基础和可供使用的亲本材料。 3. α-淀粉酶是由多基因家族编码的蛋白质，在植物种子萌发时高度表达，与植物种子的萌发能力密切相关。在大麦种子发芽时，高等电点α-淀粉酶的活性远大于低等电点的α-淀粉酶。为了研究不同穗发芽抗性青稞品种中编码高等电点α-淀粉酶Amy1 基因结构与抗性间的关系，我们以筛选得到的抗性品种‘XQ32-5’（TR1）、‘XQ37-5’（TR2）、‘XQ45-7’（TR3），易感品种‘97-15’（TS1）、‘9657’（TS2）以及强休眠大麦品种‘SAMSON’（SAM）为材料，对其Amy1 基因的编码区序列进行克隆和结构分析，并对它们推导的氨基酸序列进行比较。结果显示，青稞Amy1 基因具有三个外显子、两个内含子，编码区中有13 个核苷酸变异位点，均位于2、3 号外显子，2 个变异位点位于2 号外显子。SAM 和TS1 分别在2 号外显子相应位置有5 个相同的碱基(GAACT)的插入片段。相应α-淀粉酶氨基酸序列推导发现，所有核苷酸变异中有8 个导致相应氨基酸残基的改变，其余位点为同义突变。青稞Amy1 基因编码区序列品种间相似度高达99%以上，部分序列变异可能与其穗发芽抗性有关。随后，我们又通过SYBR Green 荧光定量技术对该基因在不同发芽时间（1d~7d）的相对表达水平进行了差异性检测。结果发现，7 天内不能检测到SAM 的Amy1 基因表达，5 个青稞品种间的Amy1 基因的相对表达量均随着发芽时间延长而上升，但上升方式有所不同。弱抗品种该基因表达更早，转录本增加速率更大，且在4~5 天可达到平台期。发芽7 天中，抗性品种总转录水平明显低于易感品种。本研究结果表明，青稞Amy1 基因的转录水平是与其穗发芽抗性高度相关。 我国青藏高原青稞，尤其是农家品种的穗发芽抗性具有丰富的变异，蕴藏着穗发芽抗性育种的宝贵资源。本研究为青稞穗发芽抗性育种建立了合理抗性评价体系，筛选出可供育种使用的特殊材料，阐明了农艺性状可辅助穗发芽抗性育种，同时还对穗发芽抗性与α-淀粉酶基因的结构和表达关系进行分析，为青稞穗发芽抗性资源筛选奠定了基础。 Preharvest sprouting (PHS) is a serious problem in crop production. It often takes place when encountering damp, cold conditions at harvest time and results in the decrease of grain quality and great loss of yield by triggering the synthesis of endosperm degrading enzymes (mostly the α-amylase). Therefore, PHS is regarded as an important criterion for crop breeding. In order to minimize the risk of PHS, resistant genotypes are highly required. Hulless barley (Hordeum vulgare ssp. vulgare) is the staple food crop in Qinghai-Tibetan Plateau from of old, where is one of the origin and genetic diversity centers of hulless barley. Recently, interest in hulless barley has been sparked throughout the world due to the demonstrations of its great potential in health food industry and fuel alcohol production. Indeed, hulless barley can also be utilized to produce good quality malt if the appropriate malting conditions are used. In China, overcast and rainy conditions often occur at maturity of hulless barley and cause an adverse on its production and application. PHS resistant genotypes, therefore, are highly required for the hulless barley breeding programs. However, few investigations have been made so far on this issue. The objectives of this study were: 1) to assessment of methods used in testing preharvest sprouting resistance in hulless barley; 2) to evaluate the variability and characteristics of PHS resistance of hulless barley from Qinghai-Tibet Plateau in China; 3) to select potential parents for PHS resistance breeding; 4) to primarily study on the molecular mechanism of PHS resistance of hulless barley. Our results are as followed: 1. We investigated the temperature effects on seed germination and seed dormancy expression of hulless barley, discussed appropriate temperature range for screening of PHS resistant varieties, and analyzed the mechanism of seed dormancy expression of hulless barley. The dormancy level of 13 hulless barley were evaluated by GI (germination index) values calculating by seed germination tests at temperature of 10，15，20，25，30℃ in darkness. There were great differences in temperature sensitivity among these accessions. The insensitive accessions showed low dormancy at any temperature while the dormancy expression of sensitive accessions could be restrained by low temperature and induced by high temperature. The temperature range of 15℃ to 25℃ was workable for estimating of dormancy level of hulless barley according to our data. Analysis of α-amylase activity showed that the temperature effects on seed germination and the expression of seed dormancy be achieved probable via regulating of α-amylase activity. Furthermore, we evaluated the differences in sprouting index (SI), sprouting rate (SR), germination index (GI) and α-amylase activity (AA) between Maerkang and Chengdu among 34 accessions of hulless barley from Qinghai-Tibetan Plateau in China. These PHS sprouting parameters were significantly affected by accession×location, and they had correlation between each other. GI was the most reliable parameter because of its low CV value, good repeatability and simple operation. SI could assist in differentiating between accessions of similar dormancy or overall evaluation of the resistance. AA was bad in repeatability and had relatively complex testing method, therefore, not appropriate for breeding and evaluation and screening of PHS resistant materials. Besides, since PHS resistance of hulless barley was greatly influenced by its growth environment, possibly much influencing factors and variations between cultivated conditions should be considered. 2. In this study, large variation was found among 113 genotypes of hulless barley (Hordeum vulgare ssp.vulgare) from Qinghai-Tibetan Plateau in China, based on the sprouting index (SI), germination index (GI) and α-amylase activity (AA) which derived from sprouting test of intact spikes, germination test of threshed seeds and determination of α-amylase activity, respectively. The range of SI, GI and AA was 1.00~8.86, 0.01~0.97 and 0.00~2.76，the mean was 4.72, 0.63 and 1.22 espectively. Six resistant genotypes, including ‘XQ9-5’, ‘XQ33-9’, ‘XQ37-5’, ‘XQ42-9’, ‘XQ45-7’ and ‘JCL’, were identified based on SI. Integrating the three parameters, it was clear that both hulls and seeds involved in PHS resistance in intact spikes of hulless barley and there was no long-existent embryo dormancy found among the test genotypes. All the genotypes, except ‘XQ45-7’, had detectable α-amylase activity on the 4th day after germination. There was PHS resistance imposed by the hull and seed per se and the two factors can act together or independent of each other. Besides, landraces or Tibet hulless barley had a wider variation and relatively more PHS resistance when compared with cultivars or Sichuan hulless barley. No significant difference was found among hulless barley of different seed colors. The correlation analysis showed PHS resistance was negatively related to hundred grain weight, days to flowering, days to maturity, spike length, awn length and flag length but not related to plant height. This study provides essential information and several donor parents for breeding of resistance to PHS. 3. Alpha-amylase isozymes are encoded by a family of multigenes. They highly express in germinating seeds and is closely related to seed germination ability. In barley germinating seeds, the activity of high pI α-amylase is much higher than low pI α-amylase. The aim of this study was to determine the relationship between preharvest sprouting resistance of hulless barley and the gene structure of Amy1 gene which encodes high pI α-amylase. The coding region and cDNA of Amy1 gene of three resistant accessions, including ‘XQ32-5’ (TR1), ‘XQ37-5’ (TR2), ‘XQ45-7’ (TR3), two susceptible accessions ‘97-15’ (TS1), ‘9657’ (TS2) and one highly dormant barley accession ‘SAMSON’ (SAM) was cloned. Analysis of their DNA sequences revealed there were three exons and two introns in Amy1 gene. Thirteen variable sites were in exon2 and exon3, 2 variable sites were in intron2. SAM and TS1 had a GAACT insert segment in the same site in intron2. Only 8 variable sites caused the change of amino acid residues. There were 99% of similarity between the tested hulless barley and some of the variable sites might be related with preharvest sprouting resistance. Then, we investigated the expression level of Amy1 gene in the 7-day germination test. Results of quantitative real-time PCR indicated that the relative expression trends of Amy1 gene were the same but had significant differences in the increase fashion between hulless barleys and no detectable expression was found in SAM. Susceptible accessions had earlier expression and faster increase and reached the maximum on day 4 ~ day 5. Besides, total transcripts level was found lower in resistant accessions than susceptible accessions. This study indicated that α-amylase activity was highly related to the transcription level of Amy1 gene which not correlated to missense mutation sites. In conclusion, hulless barley, especially the landraces from Qinghai-Tibetan Plateau in China possesses high degree of variation in PHS performance, which indicates the potential of Tibetan hulless barley as a good source for breeding of resistance to PHS. This study provides several donor parents for breeding of resistance to PHS. Our results also demonstrate that agronomic traits may be used as assistants for PHS resistance selection in hulless barley. Besides, analysis of high pI α-amylase coding gene Amy1 revealed the relative high expression of was Amy1 one of the mainly reason of different PHS resistance level in hulless barley.

~(169)Re的转动带结构组态相关性研究(英文)

研究了~(169)Re的高自旋态能级结构,建立了组态为π9/2~-[514]的强耦合带和组态为π1/2~-[541]的退耦合带,推转壳模型(Cranked shell model)计算结果表明组态相关的不同形变能够解释这些转动带的不同带交叉频率,在已知的奇ARe核中,~(169)Re的9/2~-[514]转动带在低自旋时具有最大的能量旋称劈裂,当一对i_(13/2)中子顺排后,旋称劈裂发生了反转,并且劈裂的幅度非常显著地减少了,另外,还观测到了一个三准粒子激发带,并指定了它的最可能组态为π9/2~-[514](?)νAE。

双奇核~(182)Au高自旋态能级结构及旋称反转

利用~(152)Sm(~(35)Cl,5nγ)~(182)Au核反应产生并研究了双奇核~(182)Au的高自旋态,首次建立了双奇核~(182)Au基于πh_(9/2)(×)Vi(13/2)和πi_(13/2)(×)Vi_(13/2)准粒子组态上的转动带能级纲图,发现在低自旋区,两个转动带能级均出现旋称反转.用推转壳模型对~(182)Au的转动带能级进行了理论研究,发现当采用形变和对力自洽计算后,从理论上可以定性地解释~(182)Au核中两个转动带出现的旋称反转现象。

溴的类镁离子跃迁谱线和能级扩展分析

用中国原子能科学研究院HI 13串列加速器提供的 90和 130MeV的Br离子和束箔技术 ,研究了波长在 13— 2 9nm范围内Br的类Mg离子的光谱 .识别出 2 4条属于BrXXIV的“3,3”跃迁 ,即 3s2 ,3s3p ,3p2 ,3s3d和 3p3d组态之间的跃迁谱线 ,其中 19条以前未见报道 .基于实验跃迁谱线 ,19个观测能级得以确立 ,其中有 14个能级此前未见报道 .相对论Hartree Fock(HFR)方法的计算结果对所观测的能级进行了解释 .

Prolate and oblate shape coexistence in Pt-188

A standard in-beam gamma-spectroscopy experiment for Pt-188 is performed via the Yb-176(O-18, 6n) reaction at beam energies of 88 and 95 MeV, and the level scheme for (188) Pt is established. Prolate and oblate shape coexistence has been demonstrated to occur in Pt-188 by applying the projected shell model. The rotation alignment of i(13/2) neutrons drives the yrast sequence changing suddenly from prolate to oblate shape at angular momentum 10th, indicating likely a new type of shape phase transition along the yrast line in Pt-188.

High-spin structure in Pt-187

High-spin states in Pt-187 were studied via the Yb-173(O-18, 4n) reaction. Rotational bands based on the vi(13/2), v7/2(-)[503], vi(13/2)(2)vj, v3/2(-)[512] and v1/2(-)[521] configurations were observed, and interpreted within the framework of the cranked shell model. The TRS calculations show that the vi(13/2) band has an appreciable negative gamma deformation, and the negative-parity bands tend to have a near prolate shape with small positive gamma values. Experimental values of B(M1)/B(E2) ratios have been extracted and compared with theoretical values from the semi-classical Donau and Frauendof approach, strongly suggesting a low frequency pi h(9/2) alignment in the v7/2(-)[503] band.

High-spin states in Pt-190

The level structure of Pt-190 has been studied experimentally using the Yb-176 (O-18, 4n) reaction at beam energies of 88 and 95 MeV. gamma-gamma-t coincidence measurements were carried out. Based on the analysis of gamma-gamma coincidence relationships, the level scheme of Pt-190 is extended to high-spin states. A new structure built on the 3413.6 keV 14(+) state has been observed, and the vi(13/2)(-2) vh(9/2)(-1) vj (j = p(3/2) or f(5/2)) configuration is tentatively assigned to it.

Rotation-driven prolate-to-oblate shape phase transition in W-190: A projected shell model study

A shape phase transition is demonstrated to occur in W-190 by applying the projected shell model, which goes beyond the usual mean-field approximation. Rotation alignment of neutrons in the high-j, i(13/2) orbital drives the yrast sequence of the system, changing suddenly from prolate to oblate shape at angular momentum 10h. We propose observables to test the picture.

Band properties of the transitional nucleus Pt-187

High-spin states in Pt-187 have been studied experimentally using the Yb-173(O-18, 4n) reaction at beam energies of 78 and 85 MeV. The previously known bands based on the nu i(13/2),nu 7/2(-)[503], and nu i(13/2)(2)nu j configurations have been extended to high-spin states, and new rotational bands associated with the nu 3/2(-)[512] and nu 1/2(-)[521] Nilsson orbits have been identified. The total Routhian surface calculations indicate that the transitional nucleus Pt-187 is very soft with respect to beta and gamma deformations. The band properties, such as level spacings, band crossing frequencies, alignment gains, and signature splittings, have been compared with the systematics observed in neighboring nuclei and have been interpreted within the framework of the cranked shell model. The rotational bands show different band crossing frequencies, which can be explained by the alignment either of i(13/2) neutrons or of h(9/2) protons. Importantly, evidence is presented for a pi h(9/2) alignment at very low frequency in the nu 7/2(-)[503] band. The proton nature of the band crossing is strongly suggested by comparing the measured B(M1;I -> I-1)/B(E2;I -> I-2) ratios with the theoretical values from the semiclassical Donau and Frauendof approach.

Study of quasiparticles bands in odd-odd Au-188

High-spin level structure of Au-188 has been studied via the Yb-173(F-19, 4n gamma) reaction using techniques of in-beam gamma-ray spectroscopy. Based on the experimental results, the level scheme of 188Au has been revised significantly. The previously reported positive parity levels have been modified and a new 20(+) level was proposed to feed the 18(+) states via two low-energy transitions. The existence of the 20(+) and the level structures above it are similar to those in the neighboring odd-odd Au-190,Au-192, therefore, the pi h(11/12)(-1)circle times-vi(13/2)(-2)h(9/2)(-1) configuration was assigned to the 20(+) state.

High-spin states in Au-188: Further evidence for nonaxial shape

The high-spin level structure of Au-188 has been investigated via the Yb-173(F-19,4n gamma) reaction at beam energies of 86 and 90 MeV. The previously reported level scheme has been modified and extended significantly. A new I-pi = 20(+) state associated with pi h(11/2)(-1) circle times nu i(13/2)(-2)h(9/2)(-1) configuration and two new rotational bands, one of which is built on the pi h(9/2) circle times nu i(13/2) configuration, have been identified. The prolate-to-oblate shape transition through triaxial shape has been proposed to occur around Au-188 for the pi h(9/2) circle times nu i(13/2) bands in odd-odd Au isotopes. Evidence for pi h(11/2)(-1) circle times nu i(13/2)(-1) structure of nonaxial shape with gamma < -70 degrees has been obtained by comparison with total Routhian surface and cranked-shell-model calculations.

High-spin states of odd-odd nuclei in the A similar to 170 mass region

Efforts have been made in our group to study the band structure of odd-odd nuclei in the A similar to 170 mass region. We aimed at providing new data of high-spin states and searching for the low-spin signature inversion in the 2-qp bands built on the pi h(9/2) circle times nu i(13/2) and pi i(13/2)circle times nu i(13/2) configurations. In this talk, main results of our work will be summarized, and some systematic features of signature inversion discussed. The spin and parity assignments for the pi i(13/2) circle times nu i(13/2) band in (184)An could be regarded as firm providing a good example for systematic and theoretical investigations.

~(12)C离子束辐照对酵母发酵能力的影响；Improved ethanol fermentation of a yeast mutant by C-12 ion beam irradiation

利用100MeV/u的12C6+离子束辐照酵母Saccharomyces cerevsiea YY,选育出一株高产突变菌株C03A,考察C03A发酵过程中不同温度、pH、糖汁浓度对发酵的影响。通过正交实验确定最佳发酵条件为:糖汁浓度24%、温度35℃、pH5.0。在10L发酵罐实验中,C03A发酵速率相对原始菌株高,36h发酵完全,比原始菌株缩短12h;发酵产酒率达到13.2%(V/V),比原始菌株高1.6%(V/V)。